Adherens junctions (AJs) play a fundamental role in epithelial physiology, supporting cell-cell cohesion and communication. There has been a long-standing concept that loss of functioning AJs may contribute to epithelial inflammation. In the context of the intestine, experimental disruption of the E-cadherin adhesion system induces inflammation, while E-cadherin and its regulators have been identified as susceptibility genes for inflammatory bowel disease. In this article, we briefly consider how AJ dysfunction may serve as a driver in the pathogenesis of IBD, especially in light of emerging evidence that AJs can regulate intrinsic inflammatory programs in the intestinal epithelium.
Cell-to-cell fusion, the process by which cells merge their plasma membranes to form multinucleated syncytia, is fundamental to development, physiology, and disease. Quantifying fusion in vitro typically relies on calculating the percentage of nuclei within multinucleated cells or using indirect genetic reporters. However, existing methods are laborious and error-prone, which limits standardization, accuracy, and throughput, thereby hindering meaningful mechanistic analyses. Here, we present FusionX, an AI-powered image analysis pipeline that enables automated and robust quantification of cell fusion across diverse cell types using standard membrane and nuclear dyes. FusionX integrates CellX, a fine-tuned Segment Anything Model that segments the cell boundaries of mono- and multi-nucleated cells, with Cellpose for accurate nuclear detection, enabling high-throughput and detail-rich analysis. Importantly, by extracting precise cell boundaries, FusionX provides the number of nuclei per cell together with additional single-cell parameters such as cell size and shape. Benchmarking demonstrates that FusionX delivers human-level accuracy, dramatically increases speed, and generalizes across systems, from viral fusogen-induced fusion to myogenic differentiation. By eliminating the need for specialized reporters and subjective manual quantification, FusionX paves the way for reproducible, scalable and multiparametric quantification of cell fusion in a wide range of biological contexts.
Cells migrating through three-dimensional (3D) tissues adapt their mechanical properties in response to extracellular matrix architecture through migratory plasticity. In primary human dermal fibroblasts, matrix elasticity drives distinct low- and high-pressure migration modes in which forces either push or pull the nucleus, respectively. How these mechanically distinct modes of nuclear translocation influence mitochondrial organization and function is not known. Here, we show that mitochondria become enriched anterior to the nucleus during 3D migration and segregate into spatially distinct populations with different motility and energetic states. During high-pressure, nuclear-pulling migration, a highly energized mitochondrial pool forms immediately anterior to the nucleus. This mitochondrial pool occupies a specialized perinuclear compartment organized by ROCK-dependent contractility and vimentin intermediate filaments and is selectively lost when this machinery is disrupted. Together, these findings reveal that extracellular matrix mechanics spatially organize mitochondrial dynamics and energetics during 3D migration, coupling localized mitochondrial function to the mechanical requirements of nuclear translocation. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
Biomolecular condensates have transformed our understanding of eukaryotic cellular organization, yet their roles in bacteria remain comparatively underexplored. This gap arises from the physical constraints of bacterial cell size, the diversity of molecular mechanisms that drive condensation, and the limited representation of bacterial condensates in current predictive frameworks. Here, we argue that condensation is a widespread but underrecognized principle of bacterial intracellular organization. We describe how scaffold architecture, multivalent interactions, and non-equilibrium regulation collectively determine condensate material states and cellular function. Using bacterial microcompartments as a case study, we show how diverse condensation mechanisms can converge to organize functional organelles. We further highlight emerging computational, imaging, and perturbation-based approaches that are shifting the field from condensate identification toward mechanistic understanding and predictive design. By uncovering the physical principles governing bacterial condensates, we envision their use as programmable biomaterials whose properties can be engineered to control cellular function and enable synthetic organelles.
Patient mutations within Drp1, the master regulator of mitochondrial fission, lead to severe neurological defects and poor patient outcomes. Many of these mutations have been characterized as causing functional or assembly defects in Drp1, but our study highlights three mutations (G362S, E379K, E410K) that do not have an apparent defect in core Drp1 functions. We investigated the possibility that these mutations impact interactions with Mff, a pro-fission partner protein of Drp1. Negative stain electron microscopy and mass photometry were used to visualize and quantify assembly properties, while GTPase assays assessed the enzymatic activities of distinct proteins and protein complexes. In parallel, confocal microscopy highlighted the effects of overexpressing each mutation on mitochondrial morphology in cells. We discovered that G362S and E410K Drp1 mutations limit interactions with Mff, as co-assembly into larger filaments and the associated stimulation of GTPase activity was inhibited. Conversely, the E379K mutation is able to form functional complexes with Mff, and no apparent defect was observed, warranting additional studies focused on unique mitochondrial fission attributes. Overall, our data highlight the complex nature of disease-associated mutations in Drp1 and emphasize the importance of Drp1-Mff interactions in sustaining mitochondrial and cellular health.
Proper regulation of contraction and relaxation in biological tubes is essential for organismal function. In Caenorhabditis elegans , the spermatheca, composed of smooth muscle-like cells, undergoes repeated stretching and contraction as oocytes pass through. Here we describe PES-8, a previously uncharacterized protein, as a regulator of spermathecal contractility. PES-8 contains a predicted extracellular zona pellucida-like domain and an unstructured cytoplasmic tail, suggesting dual roles in extracellular and cytoplasmic signaling. PES-8 localizes to the plasma membrane of the spermatheca, the spermathecal-uterine valve, and the uterus. Functional analysis shows that PES-8 is essential for spermathecal function; its loss disrupts actomyosin fiber alignment, FLN-1/filamin localization, apical junction organization, and Ca²⁺ signaling, preventing oocyte transit. These findings identify PES-8 as a key regulator of cytoskeletal organization and calcium-mediated contractility in the C. elegans spermatheca.
Cell line misidentification and cross-contamination remain persistent challenges in biomedical research, undermining experimental validity and contributing to irreproducible findings. Despite increased awareness and the availability of authentication technologies such as short tandem repeat (STR) profiling, problematic cell lines continue to appear in publications and commercial repositories. The International Cell Line Authentication Committee (ICLAC) maintains the Register of Misidentified Cell Lines, a curated and openly accessible resource designed to help researchers, reviewers, and funders identify cell lines that do not represent their claimed origin. The recently released Version 14 of the register expands and updates this resource with new entries and revised information. Greater awareness and consistent consultation of this resource, together with routine authentication practices, can significantly improve the reliability and reproducibility of cell-based biomedical research. Investigators are encouraged to check the ICLAC Register before acquiring or using established cell lines, authenticate cultures at defined checkpoints, including upon receipt, during master-stock generation, after extended passaging, and before publication, and report authentication evidence transparently.
Messenger RNAs (mRNAs) accumulate at centrosomes in mitosis and interphase, yet the mechanisms governing their localization and their functional significance remain poorly understood. Here, we identify a centriolar satellite - RNA-binding protein (RBP) pathway that regulates CEP350 mRNA localization and stability to support centriole overduplication. We find that CEP350 mRNA localizes to centrosomes in S phase in a microtubule (MT)-dependent manner. The RBP, UNK and centriolar satellite protein, CEP131 stabilize CEP350 mRNA and promote its steady-state levels and centrosomal protein accumulation. CEP350 is required for PLK4-induced centriole overduplication but has limited effects on canonical centriole duplication. Disrupting the centriolar satellite - RBP pathway reduces centriole overduplication in triple-negative breast cancer cells, indicating CEP131 and UNK are potential therapeutic targets for reducing centriole overduplication.
The sweeping progress in the nuclear organization-function field over the past two decades constitutes a major advance, to be sure. This work significantly impacts molecular, cell, and developmental biology and has uncovered numerous clinical implications. Here I offer perspectives around the epistemological axis in this field of ours, viz. what we knew and when, and what we know (or think we know) now.
The misfolding and aggregation of α-synuclein (α-syn), an abundant synaptic protein, leads to the pathogenesis of Parkinson's disease and related synucleinopathies. The cell-to-cell propagation of seeding-competent α-syn is initiated by unconventional protein secretion, yet the physiological pathway(s) underlying this process remain poorly defined. Here we show that α-syn secretion in human cells is mediated by Reticulon-3L (RTN3L)-dependent endoplasmic reticulum autophagy (ER-phagy), a conserved protein quality-control pathway that safeguards ER protein homeostasis. We also demonstrate that RTN3L cooperates with several autophagy regulators, including the ULK1 cofactor FIP200, to drive the delivery of α-syn into an acidic endolysosomal compartment. Increasing concentrations of α-syn disrupt ER-lysosome traffic, and α-syn-containing vesicles appear to be rerouted to the cell surface. Consistent with this proposal, knockdown of vesicle-associated SNAREs, which mediate fusion at the cell surface, disrupts α-syn secretion. These findings suggest that pathogenic α-syn secretion arises as a by-product of a physiological clearance mechanism, driven by the fusion of autophagosome-derived vesicles with the plasma membrane. Our results provide a conceptual framework for understanding how an intracellular proteostasis pathway, when misregulated, could contribute to the spread of neurodegenerative pathology.
Long-distance intracellular transport is driven by motor proteins that walk along microtubule tracks. The fate of the motor protein after transport is unclear. Classically, motor proteins have been thought to function as Diligent Workers (DW) that remain attached to cargo during the entire transport event and are degraded at the end of the journey. In contrast, previous work suggests that kinesin-1 transport can be described by a Loose Bucket Brigade (LBB) model in which individual motor proteins participate in multiple rounds of transport. Here, we used live-cell imaging in iNeurons to test whether the kinesin-3 KIF1A functions as a DW during axonal transport. We demonstrate that the fluorescence intensity of KIF1A on particles undergoing axonal transport does not change over time, suggesting that KIF1A remains attached to its cargo for the entire transport event. We determined that KIF1A has a relatively short protein half-life, consistent with KIF1A being degraded at the end of the journey. Moreover, protein turnover appears to be tightly controlled in iNeurons, as treating cells with inhibitors of the ubiquitin/proteasome system results in a cessation of KIF1A-driven transport, the appearance of KIF1A aggregates in the cell body, and their subsequent degradation through aggrephagy. These results suggest that KIF1A transport fits the DW model and that KIF1A protein levels may play a role in signaling proteostatic stress in neuronal cells.
Pulsatile cell contraction dynamics play a crucial role in tissue and cell morphogenesis. Previously, we identified a signal network in adherent mammalian cells that can transduce mechanical signals via cell contraction pulses, which are generated by fast positive feedback amplification of the signal molecule Rho via GEF-H1, and by slow negative feedback that depends on actomyosin. However, the precise mechanism was still unclear, in particular, whether it is mediated via actin or myosin-based components. Here, using numerical simulations, we predicted that network dynamics are strongly reduced both by Myosin II inhibition and by constitutive, Rho-activity-independent Myosin II activation. We confirmed these predictions experimentally by direct inhibition of Myosin II activity and by constant, non-dynamic activation via constitutively active ROCK1. Furthermore, constitutive activation of Myosin II leads to an accumulation of Myosin II next to the nuclei, which locally inhibits Rho activity dynamics. Finally, light-induced recruitment of ROCK1 to the plasma membrane strongly activated Myosin II, and at the same time depleted Actin and inhibited Rho activity. We conclude that negative feedback in the cell contraction signal network of adherent mammalian cells is implemented by Myosin II, and that actin is not the predominant inhibitory factor in this system.
Cdc14 phosphatases share conserved catalytic domains and enzymatic mechanisms. Despite functioning in disparate biological processes across eukaryotes, Cdc14 enzymes localize to analogous cellular structures from yeast to human cells. It remains unclear, however, whether modes of substrate recognition and intracellular targeting mechanisms are conserved among Cdc14 orthologs. Here, we address these questions using Clp1, the Schizosaccharomyces pombe Cdc14 phosphatase. We show that Clp1 utilizes a conserved hydrophobic pocket, originally defined in Saccharomyces cerevisiae Cdc14, to engage a subset of interactors, although unlike many S. cerevisiae Cdc14 substrates, these targets show a broad hydrophobic interaction motif spectrum. Disruption of the hydrophobic pocket selectively abrogates certain Clp1 interactions, localizations, and functions, while others are retained. Moreover, we find that nucleolar localization depends upon both hydrophobic pocket-mediated interactions and the Clp1 non-catalytic C-terminus. Our results suggest that Cdc14 uses multiple mechanisms to engage substrates and achieve proper spatial distribution for its diverse functions.
We discuss the origins of three models for the structure of chromosomes in multi-cellular animals: CTCF/cohesin loop extrusion, compartments and boundary-boundary pairing. We then review what is known about the properties of TAD (topologically associating domains) boundary elements in flies, and how these properties are deployed in generating TADs and controlling the utilization of genetic information. We discuss another class of elements that function by physically pairing with each other and their special roles in regulating gene activity. We show that TADs are organized into large TAD neighborhoods, and that neighborhoods are a common structural feature in multicellular animals. We discuss possible models for the structure of the main chromosomal axis and the underlying mechanisms for homolog/sister chromosome pairing.
Centrosome duplication is intrinsically asymmetric, creating an old and a new copy, and this difference is used to guide fate decisions during both proliferation and differentiation. The budding yeast centrosome equivalent, the spindle pole body (SPB), also duplicates asymmetrically. In yeast meiosis, this difference is used to coordinate the number of spores formed with the available carbon source. We report that the use of the older meiosis II SPBs to initiate spore formation requires a signal from acetate metabolism. This requirement is enforced by a homodimer of the Nud1 protein. Disruption of Nud1 dimerization abolishes age-dependent SPB behavior, while elevated Nud1 levels impair sporulation. We propose that dimers of Nud1 on the old SPB must be rearranged to allow meiosis-specific SPB proteins to bind and initiate spore formation. Nud1 dimerization also contributes to SPB asymmetry during mitosis, indicating that the Nud1 dimer is generally important for discriminating the age of the SPB.
Membrane lipid composition must be dynamically adjusted to preserve bilayer physical properties, yet the cellular mechanisms that support bulk lipid remodeling under physical stress remain incompletely understood. Here, we identify Csf1 as a regulator of membrane lipid remodeling functionally associated with endoplasmic reticulum-plasma membrane (ER-PM) contact sites in Saccharomyces cerevisiae, with features consistent with bridge-like lipid transfer proteins. Using high hydrostatic pressure as a defined physical perturbation that constrains membrane packing, we reveal a requirement for Csf1-dependent lipid remodeling linked to ER-PM contact sites that is masked under standard growth conditions. Quantitative lipidomic and membrane biophysical analyses show that, under hydrostatic compression, loss of Csf1 disrupts coordinated lipid remodeling, leading to reduced phospholipid unsaturation, increased membrane rigidity, and destabilization of PM permeases. We further show that Csf1 cooperates with Osh6/7 to sustain lipid flux and bilayer re-equilibration linked to ER-PM contact sites under conditions permissive for Osh6/7 activity. These findings identify Csf1 as a stress-dependent lipid-remodeling factor that enables adaptive membrane remodeling and preserves membrane protein stability under conditions of constrained membrane flexibility.
The actin cytoskeletal network is closely associated with mitochondria and performs crucial functions in mitochondrial movement, inheritance, and fission-fusion. Although its role in mitochondrial division is established, the specific contributions of actin-binding proteins (ABPs) remain unclear. Here, we report the role of tropomyosin, an ABP, in modulating mitochondrial morphology and dynamics. We demonstrate that loss of TPM1 and TPM2 in Saccharomyces cerevisiae differentially alters mitochondrial morphology. Tpm1 deletion results in fragmented mitochondria, whereas Tpm2 deletion produces an elongated tubular morphology. Through live-cell imaging, we show the localization of both paralogs to mitochondria, providing direct evidence of their association with the organelle. Microscopy-based analysis of fission-fusion frequencies revealed no change in the Tpm1 deletion, whereas Tpm2 deletion showed a decrease in these events, with a concomitant increase in the fusion factor Mgm1. Further, we characterized the overall health of mitochondria in the Tpm deletion mutants. Fragmented mitochondria in the Tpm1 deletion were hyperpolarized and exhibited increased mass and activity with elevated OCR, ATP levels, and basal ROS. In contrast, the tubular morphology of the Tpm2 deletion did not impair mitochondrial health. Overall, our findings suggest that Tpm modulates mitochondrial morphology and dynamics through its association with the actin cytoskeletal network.
Primary cilia exhibit conserved organization and contain structural and functional domains of unique molecular composition. The inversin compartment (InvC), which is found in the proximal ciliary segment of a subset of vertebrate and invertebrate cell types, concentrates different classes of signaling molecules. Mutations in genes encoding resident proteins of the InvC manifest in ciliopathies, highlighting the importance of the InvC in cilia biology. We previously showed that a chaperone of Gα proteins, RIC-8, localizes to the InvC of C. elegans channel cilia; however, the mechanisms that regulate RIC-8 targeting to this ciliary subdomain or RIC-8 function in the InvC remain unknown. Here, we build on our previous work to demonstrate that RIC-8 becomes restricted to the InvC during larval development and show that, while the RVxP motif and intact transition zone are required for its proper intraciliary distribution, RIC-8 localization to the cilium depends on intraflagellar transport. Using the ASH neuron as a model, we establish that RIC-8 modulates chemosensory responses mediated by channel cilia. Finally, we show that human RIC8A and RIC8B proteins are required for ciliogenesis in RPE-1 cells. Collectively, our results define ciliary trafficking mechanisms and novel functions for a highly conserved signaling protein.
Tetrahymena thermophila is a ciliated protist that has played pivotal roles in biological discovery. Functional studies of Tetrahymena proteins have largely relied on gene knockouts. Because protein depletion upon knockout typically spans multiple cell cycles, compensatory mechanisms can confound phenotypic interpretation. To enable rapid and acute protein depletion, we modified and adapted the trim-away system for use in Tetrahymena (Tet Trim-Away). Trim-Away is based on the E3 ubiquitin ligase TRIM21, which binds to antibody-bound proteins and targets them for proteasome-mediated degradation. Here, Trim-Away was modified with a fusion of the N-terminal RBCC (RING, B-box, coiled-coil) domains of TRIM21 with an α-mCherry (mCh) nanobody sequence that recognizes endogenously tagged mCh proteins of interest (Nb mCh ). Expression of the RBCC:Nb mCh degron, which is controlled by an inducible promoter, promotes rapid target protein depletion within 30 min and can be sustained for weeks. Tet Trim-Away is reversible, functions against targets in multiple cellular compartments, and produces loss-of-function phenotypes in Tetrahymena cells.
It has been known for over 80 years that bacterial cell size is affected by growth conditions. Cells grown in a nutrient-rich medium are larger and wider than those grown in a nutrient-poor medium. Yet even after decades of research, it is still not fully known how metabolism and cell size are coregulated. In this work, we describe a new source of metabolic control over Escherichia coli cell size, the phosphoenolpyruvate phosphotransferase system (PTS). The PTS is used to phosphorylate sugars upon entry into the cell. We found that mutations in this system result in both shorter and thinner cells and that the regulation of both dimensions of cell size appears to come from two separate mechanisms. The first mechanism regulates cell length through the production of cAMP, while the second mechanism regulates cell width through control of the levels of PEP or pyruvate in the cell.
