The glomerular capillary system is essential for efficient glomerular filtration; however, the morphogenetic processes underlying its development remain incompletely understood. In the present study, we employed two volume EM modalities—array tomography and FIB-SEM tomography—to perform 3D reconstruction of the glomerular capillary system at successive developmental stages in neonatal rats. The results revealed that glomerular capillary development proceeds through four morphologically distinct phases: primitive glomerular capillaries, initial capillary loops, maturing capillary loops, and fully matured capillary loops. Importantly, the glomerular capillary system was found to be established through the combined action of sprouting and intussusceptive angiogenesis. Sprouting angiogenesis was particularly prominent during the formation of primitive glomerular capillaries and initial capillary loops, whereas intussusceptive angiogenesis continued from the S-shaped body stage onward. Furthermore, mesangial cell processes were observed traversing transcapillary pillars and anchoring to the glomerular basement membrane, suggesting an active role for mesangial cells in capillary splitting. These findings provide the first comprehensive ultrastructural description of glomerular capillary development and offer a morphological framework for investigating glomerular capillary anomalies under pathological conditions.
Preprints have transformed how biologists share results, yet how well a preprint reads still depends on formatting, a task that has shifted from publishers to authors. Cell biology has also become increasingly quantitative, with conclusions resting on multi-step image analysis whose figures, values, and statistics are revised repeatedly across a project. Keeping a well-formatted manuscript in step with the latest analysis is tedious and error-prone. We introduce Rxiv-Maker, an open-source framework that lets authors write in simple Markdown and produce a formatted article, as a PDF or an editable Word document, with consistent styling, automatic figure and citation numbering, and reliable cross-referencing. For quantitative work, it can optionally regenerate figures and recompute reported values directly from the data, so the text stays consistent with the analysis. Built on familiar tools such as Git and Visual Studio Code, it gives a transparent, collaborative path from analysis to a submission-ready preprint. Researchers have already used Rxiv-Maker for cell-biology studies spanning bacterial cell-cycle analysis, filopodia proteomics, and zebrafish live imaging, showing it supports reproducible reporting of genuine biological findings.
Many eukaryotic cells maintain their polarity pattern and shape across multiple rounds of division. This requires that the daughter cells re-establish polarity at the correct site after cell division. Fission yeast cells are bipolar, however after division, growth initiates first at the old end of the cell that was inherited from the mother. This old-end dominance is dependent on that end having grown in the previous generation, suggesting cells must recognize when an end has a history of growth. We find that this memory of growth is imparted by the growth-dependent localization of Rax1/2 proteins. Rax1/2 promote early activation of Ras1-GTPase at dividing cell ends by recruitment of its GEF Efc25. This enables timely transition of Cdc42 activity from the division site to the old end thus giving this end an advantage in polarized growth. Consistently, modulating Ras1 activation via disruption of its regulators, Efc25 and Gap1, leads to corresponding changes in old-end dominance. Here, we demonstrate the importance of memory of growth in maintaining persistent sites of growth from one generation to the next.
LIM domain-only (LMO) proteins are associated with chromosomal translocations in T-cell acute lymphoblastic leukaemia and are highly expressed in acute myeloid leukaemia, correlating with poor prognosis. However, their role in myeloid cell lineage specification and function remains poorly understood. Using Drosophila melanogaster, which has a single LMO protein homologue, Beadex (Bx or dLMO), and a conserved myeloid-like immune system, we investigated the in vivo role of Beadex in plasmatocytes, the macrophage-like blood cells of Drosophila. Loss of Beadex reduced lymph gland size and the area of mature plasmatocytes. RNA sequencing of Beadex knockdown haemocytes revealed transcriptional changes affecting actin cytoskeleton regulation, phagosome formation and immune response pathways. Accordingly, Beadex mutant and knockdown haemocytes exhibited reduced phagocytic index and lamellipodium area. Beadex regulates actin remodelling through transcriptional control of profilin (chickadee or chic), and profilin overexpression restored the phagocytic index to control levels. Haemocyte-specific Beadex overexpression increased susceptibility to Salmonella infection, which was associated with elevated eiger (TNF) signalling and immunopathology independent of bacterial burden. Overall, Beadex coordinates myeloid cell development, cytoskeletal dynamics and immune defence, with conserved parallels in vertebrate LMO2 biology.
Prion diseases are transmissible, neurodegenerative diseases caused by misfolded, protease-resistant, and infectious aggregates of the mammalian prion protein (PrPSc) that replicate by converting properly folded prion protein (PrPC) into PrPSc. Spongiform change and cellular loss in the brain are hallmarks of prion disease, but our understanding of how prions alter cellular fitness remains incomplete. Here we characterized changes in mitochondrial redox state and respiration in neural cells following uptake of two different PrPSc strains, 22L and 87V. Only 22L PrPSc induced changes in cellular respiration and mitochondrial redox state, even in cells that did not produce PrPC. These effects were disrupted by detergent and dependent upon endo-lysosomal acidification, suggesting that both PrPSc membrane association and lysosomal degradation are involved. Interestingly, cells chronically infected with 22L appeared to adapt to infection, showing no signs of mitochondrial dysfunction, but were more susceptible to oxidative stress even though mitochondrial respiration was normal. Thus, during initial prion infection, PrPSc drives mitochondrial dysfunction in a manner that is both strain dependent and independent of PrPC expression, while persistent prion infection increases mitochondrial sensitivity to cellular stress.
Cancer cells utilize filopodia to explore, adhere to, and invade their surrounding microenvironment, yet the protein networks that organize these protrusions remain incompletely defined. To uncover the molecular machinery underlying MYO10-positive filopodia, we targeted the fast biotin ligase TurboID to the motor protein MYO10. Proximity biotinylation in two cell types revealed hundreds of potential MYO10 interactors. A targeted microscopy and siRNA screen identified MINK1, SCRIB, CSNK1A1, and CCT8 as new regulators of MYO10 filopodia formation. Focusing on one of the common interactors between cell lines, CCT8, known as a subunit of the chaperonin TRiC (TCP1 Ring Complex), we found that CCT8 associates with the MYO10 motor domain and regulates MYO10 filopodia independently of TRiC. Depleting CCT8 affected filopodia dynamics and impaired cell spreading, migration, and invasion in breast cancer cells. These findings establish CCT8 as a TRiC-independent regulator of MYO10 filopodia across diverse cancer cell types.
Stress granules (SGs) are dynamic RNA condensates that assemble rapidly in response to cellular stress following translational arrest, thereby promoting adaptation and influencing disease pathogenesis. Although SG assembly and disassembly during acute stress have been extensively characterized, their regulation under chronic stress remains poorly understood. We previously showed that chronic stress preconditioning suppresses the earliest stages of SG assembly through translation-dependent mechanisms. Whether chronic stress also impairs subsequent SG maturation has remained unknown. Here, we demonstrate that chronic stress limits SG maturation by disrupting the MYH9-dependent interaction network centered on the core SG nucleator G3BP1. Loss of this interaction reduces SG size and impairs docking between SGs and processing bodies (PBs), thereby restricting the maturation of nascent SGs. In parallel, chronic stress decreases expression of the SG nucleator UBAP2L, an essential regulator of SG-PB docking, further exacerbating these defects. Together, our findings identify chronic stress as a regulator of the MYH9-G3BP1-UBAP2L axis and reveal that chronic stress inhibits SG maturation through translation-independent mechanisms.
Transitory signaling events often hinder the identification of pathways governing cell migration and stem cell establishment during development. Using zebrafish and cultured mammalian melanocytes, we identify a previously unrecognized role for PlexinD1 signaling in neural crest-derived melanocyte lineage cells. In zebrafish, loss of PlexinD1 disrupts melanocyte migration and reduces midline melanophores derived from the regeneration-competent melanocyte stem cell (McSC) pool. In mammalian melanocytes, Semaphorin 3E-mediated PlexinD1 activation induces F-actin remodeling, altering cell polarity, protrusive dynamics, migration velocity, and directional persistence, thereby biasing migration towards attractive cues such as SCF. Beyond these acute effects, PlexinD1 activation elicits EGFR signaling and transcriptionally induces GNAS, a key effector of the MC1R pathway associated with melanocyte maturation. In zebrafish, melanocyte lineage-specific Plxnd1 ablation followed by single-cell RNA sequencing revealed altered melanocyte progenitor populations and loss of PlexinD1 activation signatures. Collectively, our findings establish PlexinD1 as a central integrator of cytoskeletal dynamics and orchestrator of signaling defining a negative chemotactic axis that is also essential for melanocyte stem cell establishment and pigment pattern formation.
Neural tube morphogenesis provides a dynamic setting to study epithelial cell behaviours. Here, we examined CLDN3-dependent biomechanical properties of the non-neural ectoderm during neural tube closure in chick embryos. Previous analyses of static images of chick embryos suggested that spinal neural tube closure occurs through multiple contact points or "buttoning". Using live imaging and transverse cuts through the neural folds we confirmed the multiple simultaneous "buttoning" contacts along the anterior-posterior axis and showed that spinal neural fold fusion is not the result of progressive "zippering" cell behaviors required in cranial and posterior neuropore regions. CLDN3-depletion decreased the rate of neural fold closure between points of contact and increased pMLC within the apical domain of non-neural ectoderm cells suggesting increased actomyosin contraction. Laser ablation studies revealed that CLDN3-depleted embryos exhibited higher tension across the non-neural ectoderm during neural fold fusion. Decreasing tension with blebbistatin rescued the neural fold fusion defects in CLDN3-depleted embryos. Our data support the importance of CLDN3 to regulate tissue tension through effects on cytoskeletal dynamics in the non-neural ectoderm during neural fold fusion.
Plasma membrane lipid asymmetry is tightly regulated and fundamental to mammalian cell physiology. TMEM30A is the β-subunit of P4-ATPases, flippase enzymes that maintain strict phosphatidylserine (PS) asymmetry by pumping it from the outer to the cytosolic leaflet. Loss of TMEM30A function causes constitutive PS externalization and has been implicated in diseases such as diffuse large B-cell lymphoma and tumor immune evasion. Here, we systematically define the biophysical and molecular consequences of TMEM30A deletion in immune cells. Using a live-cell lipid reporter, membrane order probe, and surface proteome mapping, we show that TMEM30A-knockout cells display robust PS externalization accompanied by faster lateral diffusion of membrane constituents and decreased plasma membrane order. Surface proteome reorganization includes increased abundance of tetraspanins and CD47. Furthermore, TMEM30A loss triggers glycocalyx remodeling via ADAM10-dependent shedding, which removes major transmembrane mucins, including CD43 and CD162 (also known as SPN and SELPLG, respectively). Together, these data reveal a coordinated reorganization of lipids, glycans and proteins upon TMEM30A loss, suggesting mechanistic links between flippase dysfunction and increased plasma membrane dynamics and potential sensitization to immune therapy. Furthermore, our study provides an integrated surfaceome framework that might shed light on the relationship between TMEM30A expression and clinical outcomes in cancer.
Integrating tissue-level organisation with sub-cellular resolution and molecular information often requires combining multiple microscopy modalities and scales. However, aligning images acquired with different modalities, settings, or instruments remains challenging. Here, we introduce NucleiSky, a microscopy image registration framework that utilises the spatial arrangement of nuclei or other landmarks as an intrinsic biological fingerprint. NucleiSky represents images as constellations of centroids and aligns them using geometric algorithms and spatial consensus scoring. In benchmark datasets, NucleiSky could localise query regions within larger reference images using as few as five nuclei. We show that NucleiSky can locate high-magnification fields of view within low-magnification overview scans, map these alignments to additional channels, support live brightfield-to-fixed registration using synthetic nuclear labels, and guide microscope re-targeting. We further show that the same constellation-matching principle can be extended to 3D localisation and to non-nuclear landmarks. These findings establish local landmark geometry as an intrinsic spatial fingerprint that enables localisation and registration across imaging scales, modalities and microscopy platforms. NucleiSky is available as an open-source Python package and as notebook-based applications.
Epithelial morphogenesis and homeostasis depend on dynamic remodeling of cell-cell junctions. Tricellular junctions (TCJs) control epithelial permeability and plasticity, yet how TCJs are remodeled remains unclear. In the Drosophila ovarian follicular epithelium, TCJs open transiently in a process called patency to allow passage of yolk proteins for uptake by the oocyte. Here we investigated how a TGF-β signaling gradient represses patency along the follicular epithelium. We show that TGF-β signaling blocks patency cell-autonomously by strengthening E-Cadherin (E-Cad)-based adhesion through inducing E-Cad transcription and preventing E-Cad removal from vertices. Elevated E-Cad levels alone are not sufficient to block patency, implying that additional TGF-β-dependent mechanisms stabilize E-Cad at vertices. We identify p120-catenin upregulation as a mechanism that may contribute to strengthened adhesion. In parallel, TGF-β signaling activates myosin II through Rho-Rok signaling. However, myosin II activity is dispensable for TGF-β-mediated patency suppression. Thus, our findings suggest that TGF-β signaling controls TCJ remodeling in follicle cells primarily by reinforcing E-Cad-based adhesion, disentangling the roles of adhesion and actomyosin contractility in maintaining TCJ integrity and revealing how a morphogen gradient spatially patterns epithelial permeability.
Global and community-driven initiatives have recently achieved considerable success in overcoming key challenges that hinder the widespread adoption of advanced microscopy and bioimage analysis tools in under-resourced settings. To build upon this progress, we held a workshop in May 2025 at the University of York, UK to address the needs and barriers associated with implementing time-lapse imaging and machine learning-based phenotyping in low-resource research environments. We focussed on identifying the specific challenges faced by the existing networks represented at the meeting, emphasising how integrating combined imaging hardware and machine learning-based approaches can solve these problems. This article summarises the key observations and actionable strategies made at the workshop. These proposed steps aim to significantly increase the dissemination and uptake of these powerful technologies to advance biological research in low-resource settings globally.
Oncogenic mutations of the PIK3CA gene, which encodes the catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) enhance cell migration via ERK (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) activation. We analyzed the factors regulating collective cell migration (CCM) of genome-edited MCF10A cell lines carrying hotspot PIK3CA mutations E545K or H1047R. H1047R enhanced CCM and promoted the propagation of waves of ERK activity backwards from the wound edge, whereas E545K impaired both coordinated CCM and ERK activity wave formation. The distance traveled by ERK activity waves correlated with directional persistence of migrating cells. Inhibition of cell contractility stimulated ERK wave propagation and efficient CCM of E545K cells but impaired ERK waves and CCM in control cells. Impaired ERK wave propagation was consistently associated with non-linear cell-cell junctions and the loss of polarized distribution of actomyosin. Taken together, these analyses suggest that polarized actomyosin contractility and pulsatile ERK activation must be constrained in the territory of a phase diagram compatible with mechanotransduction of ERK waves across cell-cell junctions to achieve highly coordinated and efficient collective migration.
The actin cytoskeleton is a highly dynamic and evolutionarily conserved multi-protein complex that regulates cellular architecture, mechanics and intracellular organization. Although actin dynamics and organization have been extensively studied in development and some disease contexts, the role of the actin cytoskeleton in aging has only recently begun to be considered. Accumulating evidence across model organisms indicates that aging is accompanied by progressive actin disorganization, which can manifest as filament disassembly, aggregation and mislocalization, impacting cellular homeostasis. In this Review, we synthesize the current understanding of how actin integrity is maintained through chaperone networks, actin-binding proteins, transcriptional programs and post-translational modifications, and how these regulatory layers change during the aging process. We then describe how these changes emerge as links between cytoskeletal decline and core aging hallmarks, including loss of proteostasis, mitochondrial dysfunction and cellular senescence. We further examine how this actin-related cellular dysregulation contributes to age-associated diseases, including neurodegeneration, cancer and muscle degeneration. Finally, we highlight recent studies suggesting that targeted modulation of actin regulatory pathways might preserve cellular resilience and healthspan, while emphasizing the challenges and opportunities in therapeutically targeting such a fundamental cellular system.
TRIM28, a member of the tripartite motif (TRIM) family, functions as a transcriptional coregulator involved in maintaining genome stability during mitosis. However, transcriptional activity is barely detectable during oocyte meiotic maturation. In this study, we explored the role of TRIM28 in mouse oocytes and found that it was constitutively expressed in the early stages of oocyte meiotic maturation, with predominant nuclear localization in germinal vesicle (GV)-stage oocytes. TRIM28 depletion caused defective germinal vesicle breakdown (GVBD), but oocytes that successfully underwent GVBD displayed unimpaired first polar body (PB1) extrusion. TRIM28 depletion impaired CDK1 activity and reduced cyclin B1 levels, leading to a delay in the G2/M transition. This delay may be attributed to altered levels of HDAC2-mediated H4K12ac and H3K4me2-modulated H3K9me2 in nonsurrounded nucleolus (NSN)-type GV oocytes, which decreased transcription activity. Additionally, TRIM28-depleted oocytes exhibited elevated γ-H2A.X expression, accompanied by aberrant expression of CHK1 and CHK2, as well as dysregulated expression of RAD51, which were collectively contributed to GVBD failure in mouse oocytes. In conclusion, our findings indicate that TRIM28 participates in the regulation of the G2/M transition during mouse oocyte meiotic maturation, acting through the modulation of histone modifications and DNA damage repair.
Diplonemids are highly abundant heterotrophic single-celled flagellates that are widespread in the world's ocean. They have a highly complex microtubule-based feeding apparatus (cytostome-cytopharynx complex) located adjacent to the deep flagellar pocket from which two flagella emerge. The apical papilla is a tongue-shaped structure unique to diplonemids that connects the cytopharynx and the flagellar pocket, the latter of which is formed by reinforcing microtubules (MTR) and two flagellar roots called intermediate and dorsal roots. Here we report identification of 17 proteins that localize at the feeding apparatus or flagellar apparatus in Diplonema papillatum. Using ultrastructure expansion microscopy, we show that Mad2 and its interaction partner MBP65 localize at the MTR, intermediate root, and dorsal root. Homologs of proteins that associate with the flagellar apparatus in Trypanosoma brucei (PFR1/2, KMP-11, BILBO1) localize at the feeding apparatus in D. papillatum. We also identify proteins that localize at the apical papilla, MTR, parallel microtubule loop, or cytopharynx. By discovering many components of the feeding apparatus in diplonemids, this work forms the foundation to understand the feeding mechanism in these highly abundant marine planktonic organisms.
Microtubule dynamics underpin cell division, movement, and growth in eukaryotic organisms. KATANIN p60 is a microtubule-severing protein that promotes proper cell elongation and division. In plants, division positioning is initiated in late G2 via the formation of a microtubule structure called the preprophase band (PPB). Maize p60 mutants have defects in microtubule severing and form abnormal PPBs in symmetrically dividing cells, including both incompletely assembled and misoriented PPBs. Here, we show that two types of asymmetric divisions required to generate stomatal complexes in maize p60 mutants have normally positioned but often incompletely formed PPBs. Incompletely formed PPBs lead to misoriented divisions and nuclear positioning defects in p60 mutants.
