Endometrial receptivity occurs during a limited time in the menstrual cycle called the 'window of implantation' (WOI) and is required for successful implantation. Endometrial luminal epithelial cells become adhesive to f...Endometrial receptivity occurs during a limited time in the menstrual cycle called the 'window of implantation' (WOI) and is required for successful implantation. Endometrial luminal epithelial cells become adhesive to facilitate embryo attachment and implantation; however, how this occurs is poorly understood. We recently identified that myosin heavy chain 10 (MYH10) was abnormally downregulated in infertile organoid endometrial epithelial cells during the WOI, suggesting a role in receptivity. MYH10 regulates cell polarity, adhesion and migration; however, whether it regulates receptivity is unknown. Our research investigated whether MYH10 regulates endometrial epithelial cell adhesive capacity. MYH10 is localized to all major cellular compartments within the endometrium. Immunostaining intensity was higher in luminal epithelial cells during the WOI compared to the proliferative phase in fertile endometrium. However, MYH10 staining was decreased in infertile endometrium. siRNA knockdown of MYH10 in the Ishikawa cell line significantly decreased cell adhesion to human cytotrophoblast-progenitor spheroids. MYH10 knockdown increased PGR and FOXO1 while decreasing PDLIM2 expression. Proteomics analysis following MYH10 knockdown demonstrated altered production of 57 proteins with functions critical in receptivity, including tight junctions. These results demonstrate that MYH10 alters endometrial epithelial cell adhesive capacity primarily via regulation of the actin cytoskeleton, implying an important role in implantation.
The actions of animals provide a window into how their minds work. Recent advances in deep learning are providing powerful approaches to recognize patterns of animal movement from video recordings using markerless pose e...The actions of animals provide a window into how their minds work. Recent advances in deep learning are providing powerful approaches to recognize patterns of animal movement from video recordings using markerless pose estimation models. Current methods for classifying animal behaviour using the outputs of these models often rely on species and task-specific feature engineering of trajectories, kinematics and task programming. Generalized solutions that use only pose estimations and the inherent structure of animals and their environment provide an opportunity to develop foundational, contextual and, importantly, standardized animal behaviour models for efficient and reproducible behavioural analysis. Here, we present PoseRecognition (PoseR), a behavioural classifier using spatio-temporal graph convolutional networks. We show that it can be used to classify animal behaviour quickly and accurately from pose estimations, using zebrafish larvae, Drosophila melanogaster, mice and rats as model organisms. Our easily accessible tool simplifies the behavioural analysis workflow by transforming coordinates of animal position and pose into semantic labels with speed and precision. The design of our tool ensures scalability and versatility for use across multiple species and contexts, improving the efficiency of behavioural analysis across fields.
Clonal populations exhibit phenotypic variation despite being composed of genetically identical cells under the same environmental conditions. The proliferation rate also shows this heterogeneity, but the underlying mech...Clonal populations exhibit phenotypic variation despite being composed of genetically identical cells under the same environmental conditions. The proliferation rate also shows this heterogeneity, but the underlying mechanisms remain poorly understood. In this study, we combined single-cell microencapsulation with confocal microscopy to develop a new experimental approach for analysing budding yeast cell lineages and determining the age of every cell within each microcolony. We found that most slow-growing lineages are founded by young mother cells that have undergone only a few cell divisions, typically between one and four. This reduction in proliferative capacity is linked to the expression levels of the cell cycle regulator Whi5, which increase with the number of replication cycles, even since the earliest stages. We also found that the increased levels of Whi5 are due to the higher accumulation of its mRNA during the S/G2/M phases of young mother cells compared to newborn cells. Our results show that the proliferative structure of a cell population is progressively shaped in each mitotic cycle, starting from the very first division, when a mother cell has the opportunity to establish a slowly proliferating lineage. Possible mechanisms of Whi5 action to mediate this effect are discussed.
Multicellularity emerges from the ability of cells to undergo functional differentiation. One of the key mechanisms that enables this coordination is cellular signalling-a series of molecular interactions within or betwe...Multicellularity emerges from the ability of cells to undergo functional differentiation. One of the key mechanisms that enables this coordination is cellular signalling-a series of molecular interactions within or between cells that induce changes in cell behaviour or gene expression. As the body plan of multicellular organisms becomes more complex, so does the sophistication of their signalling systems. The Wnt and Notch pathways are central to regulating cell fate, tissue development and maintenance in all studied metazoa. Affecting overlapping biological processes, often within short developmental time windows, these molecular systems appear to be functionally interconnected, leading to the proposal of a 'Wntch' signalling concept. This concept implies that Wnt and Notch modules do not operate as isolated linear pathways but form a coherent network that integrates signals to ensure precise control of developmental and physiological outcomes. In this review, we synthesize both past and recent insights into the direct crosstalk of Wnt and Notch signalling molecules, examine crosstalk within the context of recently developed assays such as single-cell RNA sequencing and proximity labelling, and discuss the broader implications of this interplay in development and disease.
Synaptonemal complex (SC) is a structurally conserved, supramolecular assembly that forms at the interface of aligned chromosome axes during meiosis, where it provides a physical context for crossover recombination inter...Synaptonemal complex (SC) is a structurally conserved, supramolecular assembly that forms at the interface of aligned chromosome axes during meiosis, where it provides a physical context for crossover recombination intermediates. In yeast, the SC is composed of Zip1 transverse filaments and central element proteins Ecm11 and Gmc2. Here, we identify a biochemically stable constitutive complex between Ecm11 and Gmc2, which is mediated by their α-helical coiled-coil regions formed of amino acids 230-302 and 59-188, respectively. We find that the Ecm11-Gmc2 is a 2 : 2 hetero-oligomer, which has an architecture and dimensions similar to the mammalian SC central element complex SYCE2-TEX12. Through targeted mutagenesis in yeast, we show that 2 : 2 Ecm11-Gmc2 complex formation is essential for SC assembly in vivo. Further, we identify key additional residues, particularly in Ecm11, that are dispensable for heterocomplex formation in vitro but critical for stability of the complex in vivo.
Siegel A, Almstead D, Kothandaraman N
… +15 more, Reich J, Lamkin E, Victor J, Grover A, Ikeh K, Koval H, Crompton A, Jang H, Lee H, Del Rio-Guerra R, Korzhnev D, Hadden MK, Hong J, Zhou P, Chatterjee N
Trinucleotide repeat instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. While germline expansions destabilize trinucleotide repeats to cause disease anticipation, somatic cell tr...Trinucleotide repeat instability has been implicated in the pathogenesis of numerous neurodegenerative disorders. While germline expansions destabilize trinucleotide repeats to cause disease anticipation, somatic cell trinucleotide repeat instability drives earlier onset of symptoms and further disease progression. However, the drivers behind these repeat length changes remain unclear. Current models suggest that DNA replication slippage events and the action of genome instability pathways, such as DNA repair, cause trinucleotide repeat mutagenesis. Whether mutagenic polymerases from the translesion synthesis pathway result in trinucleotide repeat instability is unclear. Translesion synthesis polymerases are best at bypassing difficult-to-replicate DNA regions due to bulky lesions or gaps in DNA. While some effects of translesion synthesis polymerases on trinucleotide repeat instability have been explored in lower organisms, evidence in human cells is lacking. Using a quantitative green fluorescent protein (GFP) reporter with expanded CAG repeats, we show that inhibition of the translesion synthesis polymerase REV1 by its inhibitor, JH-RE-06, or siRNA knockdown increases trinucleotide repeat instability and the underlying mutability. These results suggest that REV1 protects trinucleotide repeat length mutagenesis through potential continuous DNA synthesis when replicative polymerases stall ahead of repeat secondary structures. Collectively, we present evidence of the translesion synthesis pathway's role in trinucleotide repeat instability, with potential implications for understanding mutability mechanisms, disease biology and therapeutic targeting.
Bioelectrical signalling is fundamental for regulating biological processes in all forms of life. Ion channels and transporters generate and propagate electrical currents by selectively allowing ions to flow across membr...Bioelectrical signalling is fundamental for regulating biological processes in all forms of life. Ion channels and transporters generate and propagate electrical currents by selectively allowing ions to flow across membranes in response to voltage changes. Although recent breakthroughs in structural determination methods, such as cryogenic electron microscopy, have provided novel insights into the structure-function relationships of ion channels and scaffolding proteins, their precise roles in bioelectrical signal generation and propagation within and across different cells and tissues remain unresolved. This article examines the biochemical and ultrastructural features of the three most studied modes of bioelectrical conduction in human tissues-electrotonic, saltatory and ephaptic conduction-and how biophysical constraints set by membranes and proteins give rise to bioelectricity. Notably, ion channel clustering and scaffolding proteins that define intermembrane distances are common key features among all forms of bioelectrical signalling. Techniques like cryogenic electron tomography offer promising avenues for exploring ion channels and their regulatory protein interactions in situ. The central question is: 'How does the spatial organization of ions, molecules and tissues give rise to bioelectricity?' These insights may inform novel therapeutic approaches for various diseases, while also potentially offering new perspectives on life, evolution and consciousness.
The SWI/SNF family of chromatin remodelling complexes, comprising BAF, PBAF and ncBAF, is known for their critical roles in regulating chromatin accessibility and gene expression in mammalian cells. Recent advances have...The SWI/SNF family of chromatin remodelling complexes, comprising BAF, PBAF and ncBAF, is known for their critical roles in regulating chromatin accessibility and gene expression in mammalian cells. Recent advances have shed light on a function for SWI/SNF complexes, particularly PBAF, at centromeres. In this review, we explore the emerging roles of SWI/SNF complexes in safeguarding centromere stability and discuss how disruption of PBAF leads to centromere fragility. We propose that PBAF contributes to the establishment and maintenance of boundaries between heterochromatin and euchromatin regions within centromeres and pericentromeres, thus contributing to their overall architecture. By preserving these boundaries, PBAF ensures the functional integrity of centromeres, which is essential for faithful chromosome segregation.
Characterizing the effect of pesticides on pollinators is essential in the strive to protect biodiversity while maintaining efficient food production. Metabolomics offers detailed insight into the physiological response...Characterizing the effect of pesticides on pollinators is essential in the strive to protect biodiversity while maintaining efficient food production. Metabolomics offers detailed insight into the physiological response to pesticides. The impact of pre-dissection and dissection methodology on the metabolic response remains largely unknown, as does their possible effect on the measured metabolic response to pesticide exposure. Three different pre-dissection treatments were evaluated in Eristalis tenax: carbon dioxide, ice or no anaesthesia. Brain dissections were conducted at room temperature or on ice. Flies were also orally exposed to a high dose of the neonicotinoid insecticide acetamiprid (4 μg per fly) in sucrose or sucrose alone. Brains were homogenized, and metabolites extracted and analysed by gas chromatography/mass spectrometry. Pre-dissection and dissection conditions affected metabolites linked to oxidative stress, energy production and cold response. Acetamiprid exposure elicited consistent metabolic responses across all immobilization methods, including significant alterations in glutamate metabolism. Alterations in brain metabolism in response to acetamiprid were largely conserved across various pre-dissection methods, allowing for flexibility in methodology to address experimental constraints. Whether the subtle differences observed would compromise studies of lower doses of acetamiprid or other pesticides requires further validation.
Ionizing radiation (IR) is used to treat more than half of cancer patients because it induces DNA double-strand breaks and triggers apoptosis. IR also damages other nucleic acids, lipids, proteins and cellular organelles...Ionizing radiation (IR) is used to treat more than half of cancer patients because it induces DNA double-strand breaks and triggers apoptosis. IR also damages other nucleic acids, lipids, proteins and cellular organelles, initiating additional complex cellular responses. Some of these responses are transient, while others can become permanent and lead to changes in cellular identity. This review focuses on cell fate plasticity, defined as the conversion of one cell type into another, during recovery after IR-induced damage. We recognize that this process likely occurs along a continuum and may be reversible. We will distinguish cell fate plasticity from molecular or phenotypic plasticity, such as epigenetic modifications, transcriptomic shifts, altered signalling, morphological changes or acquisition of migratory behaviour, all of which are clinically relevant but do not constitute a change in cell type for the purposes of this review. Importantly, cell fate plasticity can enable cancer cells to acquire stem-like properties, which has major implications for tumour progression and therapy resistance.
SACK1G (aka FAM83G, PAWS1) plays a central role in activating canonical WNT signalling through interaction with the Ser/Thr kinase CK1α. The loss of CK1α binding and WNT signalling underlies the pathogenesis of palmoplan...SACK1G (aka FAM83G, PAWS1) plays a central role in activating canonical WNT signalling through interaction with the Ser/Thr kinase CK1α. The loss of CK1α binding and WNT signalling underlies the pathogenesis of palmoplantar keratoderma (PPK) caused by several reported mutations in the SACK1G gene. We modelled the scaffold anchor of CK1 (SACK1) domain of SACK1G and used fragment-bound structures of the SACK1B (FAM83B) dimer to guide our analysis. This allowed us to computationally predict several key residues near the fragment-binding site in SACK1G that may be important for its function. We mutated these residues, introduced them into SACK1G-/- DLD-1 colorectal cancer cells and investigated their ability to bind endogenous CK1α. We uncovered two SACK1G mutations, namely Y204A and I206A, that abolish interaction with CK1α similarly to the PPK pathogenic mutant A34E. Consistent with this loss of SACK1G-CK1α interaction, the molecular glue degrader of CK1α, DEG-77, fails to co-degrade the Y204A and I206A mutants while it still co-degrades native SACK1G. Our findings demonstrate the utility of our computational methods to uncover functional residues on proteins based on fragment-binding sites.
The glial cells missing (GCM) genes were first discovered in Drosophila and encode transcription factors important for gliogenesis. In placental mammals, GCM1 regulates several genes that are important for early placenta...The glial cells missing (GCM) genes were first discovered in Drosophila and encode transcription factors important for gliogenesis. In placental mammals, GCM1 regulates several genes that are important for early placenta development, while its paralogue GCM2 is important for parathyroid gland development. The egg-laying monotremes, which represent the most diverged extant mammalian lineage, undergo a short period of intrauterine development and form a simple placenta. To gain more insight into the evolution of GCM genes, we analysed the sequence, expression and genomic localization of GCM1 and GCM2 genes in the platypus and echidna. We found that the chromosomal localization of GCM1 changed after the divergence of therian mammals, coinciding with the evolution of a complex placenta. Expression analysis revealed the presence of GCM transcripts in male and female monotreme gonads, as well as expression of GCM1 in the female reproductive tract. GCM-binding sites in target genes associated with placental development in therian mammals were also present in the monotremes and the chicken. Together, this suggests that the role of GCM1 in reproduction emerged early in mammalian evolution.
Chronic pain-the persistence of pain-delineates a huge challenge to the healthcare system. During the process of pain chronification, when acute pain transitions into chronic pain, processing and pathways associated with...Chronic pain-the persistence of pain-delineates a huge challenge to the healthcare system. During the process of pain chronification, when acute pain transitions into chronic pain, processing and pathways associated with nociceptive impulse progressively undergo diverse anatomical and physiological alterations in a process known as sensitization. Pain sensitization, which occurs in both the peripheral and central nervous system, entails intricate neurobiological mechanisms that can lead to a cascade of effects, including reduced pain threshold, augmented excitability of pain pathways and heightened sensitivity to painful stimuli. Due to the complex interplay of these neurobiological mechanisms, optimal treatment for chronic pain remains elusive. Elucidating these mechanisms further is essential to devise targeted interventions for preventing or managing chronic pain, particularly following surgical procedures. This review explores the key neurobiological mechanisms involved in the transmission of nociceptive signalling, an in-depth mechanism contributing to pain chronification, with a special focus on peripheral and central sensitization, neuroimmune interactions and neuroplastic changes within the central nervous system. It also encompasses a comprehensive overview of various therapeutic interventions, which is crucial for enhancing therapeutic strategies and patient outcomes in chronic pain management.
Trehalose accumulation is commonly observed in extremophile organisms capable of surviving extended periods of complete desiccation and freezing. However, direct evidence linking the sugar to cryptobiotic survival in met...Trehalose accumulation is commonly observed in extremophile organisms capable of surviving extended periods of complete desiccation and freezing. However, direct evidence linking the sugar to cryptobiotic survival in metazoans is generally lacking. Here, using the extremophile crustacean Artemia franciscana as a model, we test the anhydrobiotic and cryobiotic roles of trehalose through RNAi-mediated knockdown of its biosynthetic and transport pathways. Transcriptomic and genomic screening uncovered a major expansion of trehalose transporter (tret) genes in branchiopods with up to 27 copies in Artemia sp., but only a single trehalase enzyme (treh), and three trehalose-6-phosphate synthase (tps) genes containing fused synthase and phosphatase domains. Gene expression analysis determined that the tps and selected tret genes are expressed within the diapause-programmed oocytes and embryos, with independent RNAi-mediated knockdown of each reducing cyst trehalose accumulation by approximately 90% and 30%, respectively. Trehalose depletion in diapause cysts critically affects the anhydrobiotic and cryobiotic survival of embryos, a feature confirmed through diapause-termination experiments using either desiccation or H2O2 exposure. These latter data further uncovered the developmental reprogramming of carbohydrate metabolism to cope with low amounts of stored trehalose in the cysts. The findings demonstrate that diapause-induced biosynthesis and transport of trehalose are essential for A. franciscana anhydrobiosis.
Excitatory amino acid transporters not only mediate high-affinity glutamate uptake but also conduct an uncoupled chloride current. In zebrafish, a whole-genome duplication gave rise to two paralogues with distinct roles...Excitatory amino acid transporters not only mediate high-affinity glutamate uptake but also conduct an uncoupled chloride current. In zebrafish, a whole-genome duplication gave rise to two paralogues with distinct roles. Excitatory amino acid transporter 2a (SLC1A2b, GLT-1) functions primarily in Müller glia as a glutamate transporter, whereas excitatory amino acid transporter 2b is expressed in cone photoreceptors and exhibits a prominent glutamate-independent chloride current. We hypothesized that this leak current stabilizes the cone resting membrane potential, thereby supporting rapid visual signalling. In order to test this hypothesis, we generated knockout zebrafish using CRISPR-Cas9-mediated genome editing. While mutants showed no gross morphological abnormalities, they exhibited reduced electroretinogram b-wave amplitudes. Consistent with our hypothesis, -deficient larvae displayed a significant reduction in flicker fusion electroretinogram power at each stimulus frequency, indicating impaired temporal processing likely due to delayed repolarization of cone photoreceptors. Our findings reveal a critical role for an excitatory amino acid transporter 2b-mediated chloride anion leak current in regulating the kinetics of photoreceptor responses. This functional innovation, enabled by a whole-genome duplication in the teleost lineage, highlights how gene duplications can lead to the acquisition of physiologically relevant new functions.
Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in sl...Although the difference between the characteristics of fast and slow muscles has been extensively studied, it is still not fully understood. Here, we propose that nicotinic acetylcholine receptors (AChRs) expressed in slow muscles of zebrafish have high Ca permeability compared to that of AChRs of fast muscles. To analyse the significance of the Ca influx through AChRs in slow muscles, we generated a transgenic (Tg) zebrafish line that expresses Ca-impermeable AChRs in its slow muscles. The locomotor activities of the Tg zebrafish were markedly decreased at 1-3 days post-fertilization (dpf) compared to those of zebrafish expressing Ca-permeable AChRs in their slow muscles. Ca imaging suggested that Ca influx via AChRs is crucial for the Ca response during muscle contraction in 2 dpf larvae, as slow muscle cells of the Tg line lacked a sustained Ca response. Furthermore, we found that slow muscles of the Tg line became thinner compared to those expressing Ca-permeable AChRs. These short Ca responses and thinner slow muscles may have induced locomotion impairment in the Tg line. These results suggested the physiological roles of the Ca influx through AChRs in slow muscles and provided new insights into the characterization of fast and slow muscles.
Neuronal morphogenesis is regulated by intracellular transport and cytoskeletal dynamics. Kinesin superfamily proteins (KIFs), or kinesins, function as molecular motors for intracellular transport and as regulators of th...Neuronal morphogenesis is regulated by intracellular transport and cytoskeletal dynamics. Kinesin superfamily proteins (KIFs), or kinesins, function as molecular motors for intracellular transport and as regulators of the microtubule cytoskeleton, making them essential for neuronal development. has been widely used as a model organism to study neuronal morphogenesis. Due to the critical roles of kinesins in neuronal functions, numerous kinesin mutants, including unique gain-of-function mutants and temperature-sensitive mutants, have been identified through forward genetic screens in . The availability of whole-genome knockout resources and CRISPR/Cas9 genome editing has further enabled precise genetic analysis, facilitating the modelling of human kinesin-related diseases in . In this review, we discuss the functions of kinesins in neuronal morphogenesis, focusing on their roles in neuronal transport and cytoskeletal regulations.
The specification of abdominal segments A5 to A9 depends on the expression of (), which is regulated by four domains: through . Each domain contains an initiation element that determines its active state, along with...The specification of abdominal segments A5 to A9 depends on the expression of (), which is regulated by four domains: through . Each domain contains an initiation element that determines its active state, along with enhancers responsible for tissue-specific activation of . These domains function autonomously due to their flanking boundaries, , and , which both block crosstalk between adjacent domains (insulator function) and facilitate long-range interactions with the promoter (bypass function). In inactive domains, enhancers are repressed by Polycomb group (PcG) proteins. Activation of the domains is driven by initiators, which are stimulated in a segment-specific manner by products of the gap and pair-rule genes during early embryogenesis. By creating truncations of the boundary, we identified that the bypass module is adjacent to CTCF binding sites and overlaps with sequences responsible for recruiting PcG proteins. Using genome editing, we demonstrated that the and initiators enhance the activity of the bypass module. Therefore, initiators represent a novel class of regulatory elements that control long-distance interactions between enhancers and promoters.