How does the cell coordinate its two major activities of cell growth and cell division? To explore this, we have genetically depleted yeast ribosomal protein Rpl32 of the 60S ribosomal subunit, which is an essential protein for cell proliferation. After 3-4 h of Rpl32 depletion, the cell cycle arrests at G1. We have undertaken a kinetic analysis of the early cellular events to deduce the pathway from Rpl32 depletion to G1 arrest. Rpl32 depletion blocks pre-rRNA processing of the initial 35S pre-rRNA, thus preventing ribosomal biogenesis and nuclear export of 60S ribosomal subunits. Interestingly, the Rpl25-GFP reporter transiently accumulates in a focal spot that resembles the nucleolar body/Cajal body. Amazingly, the inhibition of ribosome biogenesis in the nucleus is signalled to the cytoplasm, where mature 18S and 25S rRNAs are degraded in a ribophagy-independent manner; Rpl32 protease-degradation uses de-ubiquitination. Nonetheless, the ribosomes that remain after degradation are sufficient for translation, whose efficiency is unchanged through 6 h after Rpl32 depletion, and the cell size and vacuole increase in size. The level of cyclin 1 mRNA is rapidly diminished after Rpl32 depletion and is a likely factor for the arrest of the cell cycle at G1.
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.
Actin bundle-supported membrane protrusions contribute to a range of physiological functions across cell types. An interesting case is the tuft cell, which assembles exaggerated microvillus-like protrusions that support chemosensory functions in the intestinal tract. Tuft cell protrusions are supported by large core actin bundles; several F-actin bundlers are expressed in tuft cells, and these exhibit regionalized localization along the core bundle axis. Uniquely, LIM domain and actin binding 1 (LIMA1) is restricted to the basal, rootlet ends of these bundles. How LIMA1 impacts protrusion formation and core bundle architecture remains unclear. Here, we leveraged multiple forms of microscopy to examine the impact of LIMA1 on the actin cytoskeleton in epithelial and nonepithelial models. Our findings indicate that LIMA1 expression is sufficient to drive the elongation of microvilli in epithelial cells, as well as the formation and stabilization of exaggerated filopodia in a non-epithelial context. Importantly, we found that individual filopodia in LIMA1-overexpressing cells exhibit merging dynamics, which enable the formation of large core bundles from smaller precursors. These data indicate that LIMA1 exerts potent effects on rootlet actin architecture and is well positioned to support the formation and maintenance of large core actin bundles, like those assembled by tuft cells.
Membrane tethering is essential for the generation of organelle contact sites, the catabolic process of autophagy, and to anchor incoming vesicles to their target membranes before vesicle fusion. Although membrane tethering is critical for cellular function, many of the current biochemical techniques to test for membrane tethering rely on indirect readouts and are limited in their ability to monitor protein localization at sites of tethering. As such, we recently developed a fluorescence microscopy-based giant unilamellar vesicle and liposome tethering assay (GLT) to study the membrane tethering properties of two autophagy proteins. In this study, we used GLT with engineered membrane tethers to demonstrate the ease of use, methods of analysis, versatility, and sensitivity of the assay. We demonstrate that: 1) GLT can be used to study liposome tethering, fusion, and phosphatase-mediated detethering of tethered liposomes, 2) GLT detects tethering with comparable sensitivity with less direct methods for monitoring membrane tethering while allowing simultaneous monitoring of membrane and protein localization, and 3) GLT can be used to monitor the kinetics of membrane tethering in real time. Collectively, our results demonstrate GLT is a broadly useful method to study membrane tethering in vitro.
Iron overload cardiomyopathy (IOC) is caused by elevated systemic iron, and it is characterized by systolic and diastolic dysfunction as well as arrhythmias. Isolating the cardiac-specific cellular and molecular mechanisms driving IOC has been challenging because it affects multiple interconnected organ systems. Here, we leverage stem cells, cardiac tissue engineering, and protein reconstitution to model key contractile aspects of human IOC in vitro and probe the cellular and molecular mechanisms driving cardiac dysfunction. Human-engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced contractile function, impaired relaxation, and increased prevalence of arrhythmogenic events. While both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, cardiomyocytes show higher iron accumulation and reactive oxygen species production. Moreover, iron overload has little effect on the action potential kinetics in engineered heart tissues; however, it impacts the kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and iron-based inhibition of myosin. Our results reveal insights into the cellular and molecular mechanisms of human IOC pathogenesis and establish in vitro models that can be harnessed for mechanistic and translational studies.
Cell elongation and cytokinesis are mediated by two large multi-protein complexes in rod-shaped bacteria: the elongasome and divisome. These membrane-associated complexes ultimately recruit peptidoglycan cell wall remodeling and synthesis machineries, thus sculpting and extending growing cells or driving septation of two daughter cells. The elongasome and divisome have been well studied in model bacterial species, including Bacillus subtilis, Caulobacter crescentus, and Escherichia coli. Here, we present an analysis of these complexes in an obligate intracellular bacterium with a highly reduced genome. Orientia tsutsugamushi, a cytoplasm-dwelling Gram-negative alphaproteobacterium, only retains a subset of proteins normally found in the elongasome and divisome. It also lacks all copies of the major peptidoglycan polymerase, Class A Penicillin Binding Protein, and has been shown to build an intermediate peptidoglycan cell wall-like structure that is low in abundance and insufficient to consistently confer a rod shape to the bacterium. We have carried out a first analysis of the elongasome and divisome in Orientia tsutsugamushi, quantifying the expression and subcellular localization of five key proteins through early stages of the intracellular infection cycle. We show how these are affected by antibiotic treatment and present a model for minimal elongation and division in an obligate intracellular bacterium.
The SAF-A/HNRNPU gene encodes an abundant nuclear protein conserved throughout vertebrates, and is mutated in individuals with HNRNPU syndrome, a neurological human disease. SAF-A is important for maintaining lncRNA localization, splicing, and gene expression state. The mechanistic role of SAF-A in each of these processes is likely coordinated by one or more of its functional domains, which include an N-terminal SAP domain, a central ATPase domain, and an RGG domain defined by a series of C-terminal RGG/RG repeats embedded within a low-complexity region. However, a comprehensive analysis to identify which SAF-A domains are required for each cellular function is lacking. Here, we use an allelic reconstitution strategy to investigate the role of the SAF-A ATPase and RGG domains in lncRNA localization, nuclear dynamics, transcription, splicing, and cell viability. We show that both the ATPase and RGG domains control SAF-A nuclear dynamics, and that SAF-A interacts with nascent RNA Pol II transcripts through the RGG domain. The SAF-A ATPase and RGG domains were required for maintaining XIST RNA and facultative heterochromatin marks on the inactive X chromosome, but did not affect X-linked gene silencing. The SAF-A ATPase and RGG domains were both required for proper mRNA splicing, but not for gene expression. Importantly, we found that the SAF-A ATPase and RGG domains were required for cell proliferation, arguing that these domains are each linked to the essential cellular functions of SAF-A. Collectively, our findings highlight the importance of the SAF-A SAP, ATPase, and RGG domains in vital functions of nuclear biology.
In diderm bacteria, the outer membrane (OM) must invaginate in concert with septal peptidoglycan (PG) remodeling during cytokinesis. One OM lipoprotein, DolP, has been shown to localize at the cell division site and facilitate the daughter cell separation. Yet how DolP is recruited remains unclear at the molecular level. Here, we show that DolP arrives at mid-cell concomitantly with the late divisome protein FtsN. Utilizing single-particle tracking Photoactivated Localization Microscopy (spt-PALM), we investigated the dynamics of individual DolP molecules in living Escherichia coli cells. Single-molecule analysis revealed two diffusion states: a diffusive state across the cell envelope and an immobile state enriched at the septal and polar regions. Because anionic phospholipids are known to be enriched at regions of high negative curvature, we tested mutations in the DolP anionic phospholipid-binding surface and found they abolished mid-cell enrichment and reduced the immobile fraction. Importantly, DolP's localization is independent of division proteins like EnvC and NlpD, and DolP does not comigrate with the core septal synthesis complex FtsW-FtsI-FtsN complex. Instead, DolP enrichment requires an actively constricting septum. Together, these findings support a model in which anionic phospholipid-mediated diffusion-state switching drives DolP enrichment at the division site.
Wound repair is an essential biological process that occurs both in tissues and single cells. In free-living single-celled ciliates such as Stentor coeruleus, rapid repair from wounds is necessary to heal breaches to the plasma membrane, where any delays represent the difference between life and death. In order to discover novel molecular pathways that are important for healing in Stentor, we carried out a targeted RNA interference-based perturbation genetic screen combined with microsurgical wounding using a microfluidic guillotine to introduce reproducible bisection wounds. We identified a Stentor syntaxin gene that was necessary for cell survival, particularly post-wounding, with only ∼37% of syntaxin-deficient cells surviving compared with ∼98% of control cells. Syntaxin-deficient cells were more susceptible to hypoosmotic shock and became increasingly vacuolated in the hours post-wounding, eventually leading to cell death. Wounding of the cells in 75 mM sorbitol during and after bisection partially restored the post-wound survival in knockdown cells. These results support the interpretation that syntaxin-deficient cells lack essential membrane fusion machinery, which manifests in vacuolar defects, and are deficient in maintaining osmotic homeostasis necessary for their survival post-wounding. This study provides a template for the discovery of new wound healing biology in emerging model systems.
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.
Epithelial wound healing is an essential process in multicellular organisms, primarily driven by lamellipodia-based crawling, purse-string contraction, and collective cell migration. One or more of these mechanisms participate in healing a wound, yet the choice, sequence, and coordination of these processes are poorly understood. Moreover, different mechanisms dominate in different tissues, organisms, wound types, and wound sizes, further complicating our understanding of how cells select among healing mechanisms. In this study, we analyzed wound healing across wound types and spatial scales in the basal eukaryote Clytia hemisphaerica (Clytia) to establish a unified model for mechanism selection within a single organism. We demonstrate that lamellipodial crawling and actomyosin cable contractions are sequential, partially redundant processes involved in healing all wounds. Furthermore, the exposure of the basement membrane acts as a central regulatory cue, orchestrating lamellipodia formation, actomyosin contraction, and collective cell migration responses. Remarkably, we discovered that these same mechanisms operate in healing micro-wounds internal to a single cell. This work fundamentally advances our understanding of how diverse healing mechanisms are coordinated to respond to all types of wounds, while the use of a basal metazoan model expands our knowledge of fundamental strategies for maintaining epithelial integrity.
Collective cell migration is central in development and disease. Vimentin is an intermediate filament protein expressed by epithelial cells at the edge of wounds where collective cell migration is most efficient. Yet, its functional role in this context remains underexplored. Here, we show that vimentin, over-expressed in cells undergoing partial epithelial to mesenchymal transition at the edge of epithelial monolayers, has a multiscale impact on the whole monolayer mechano-dynamics. Vimentin knock-down delays wound closure, reduces cell coordination, while increasing traction forces exerted by cells on the substratum. It also disrupts the directionality of leader cells migration, as well as the cohesion and coordinated motion of cells deep in the monolayer. We further show that vimentin promotes the conversion of polarized cell locomotion into coordinate collective migration by polarizing actin, focal adhesions and traction forces, sustaining leader cell's lamellipodium protrusive activity and directionality, while allowing mechanical coupling of leader with follower cells. Altogether, we show that vimentin is essential for bridging polarized single cell locomotion and coordinated collective migration to allow efficient collective migration. [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] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
The light-responsive outer segment of rod photoreceptors is composed of two distinct membrane subdomains: discs and the plasma membrane. We investigate how the disk protein peripherin-2 is engaged in CNG channel delivery to the outer segment. Instead of forming outer segments, peripherin-2 knockout (Rds-/-) photoreceptors release ciliary ectosomes, in which CNG channel levels are markedly reduced relative to other outer segment proteins. This is intriguing as downregulation of the CNG channel is not a general feature of degenerative mouse models with dysmorphic outer segments. Overexpression of the β1-subunit of CNG in Rds-/- rods reveals that the majority is trapped in intracellular membranes, but is restored to the outer segment by co-expressing peripherin-2. We test peripherin-2 chimeras containing either the N-terminus, tetraspanin core, or C-terminus and find that the tetraspanin domain is sufficient to localize CNGβ1 to the outer segment. We further show that the membrane remodeling function of the tetraspanin domain facilitates this process by the redundant action of the tetraspanin domain from ROM1 and the reemergence of the endogenous CNG channel in aged Rds-/- rods that have produced ciliary membrane protrusions.
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. [Media: see text].
Animal tissues have diverse architectures and cell behaviors across the epithelial-mesenchymal spectrum. Cell adhesion mediated by classical cadherins is foundational. Cadherins nucleate complexes of dozens of proteins connecting junctions to the cytoskeleton and signaling downstream. Many junctional proteins are well-studied in epithelia but less is known about roles during mesenchymal migration. The nascent myotubes of the pupal Drosophila testis provide an excellent model for N-cadherin mediated mesenchymal migration. We combined a proximity proteomics dataset of adherens junction proteins in mammalian epithelial cells with genome-wide shRNA libraries knocking down Drosophila genes to begin to define the subset of junctional proteins important in mesenchymal migration. While N-cadherin is predominant, E-cadherin plays a supporting role. Surprisingly, several proteins with key roles in epithelial morphogenesis, including Afadin's homolog Canoe, ZO-1's homolog Polychaetoid, and Par3's homolog Bazooka play at most modest roles. Twenty-two genes with diverse cell biological roles had strong to moderate defects in testis morphogenesis. These will provide a community resource. We followed up two. The kinase Par-1 is important for migration and gap closure, with knockdown phenotypes paralleling those of myosin. The Rab GAP RN-tre does not have roles until after migration and works in parallel with N-cadherin during testis spiralization. [Media: see text] [Media: see text].
The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast Saccharomyces cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Cdh1 degradation is mediated by the APC/C itself in a "trans" mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. Degradation requires an intact glucose-sensing SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which directly phosphorylates Cdh1 on Ser-200 within an unstructured N-terminal region. In the absence of phosphorylation, expression of a Cdh1-S200A mutant is fully stabilized, leading to defective meiosis or spore formation and loss of viability. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.
T cell activation induces a rapid reorganization of the actin cytoskeleton, facilitating cell spreading on antigen-presenting cells (APCs). As the nucleus, the cell's largest organelle, constrains cell shape changes, its mechanical properties, governed by chromatin compaction and heterochromatin density, are likely to play a critical role in T cell spreading and activation. However, the contribution of nuclear mechanics to T cell activation remains elusive. Here, we demonstrate that T cell spreading is accompanied by nuclear deformation and increased chromatin compaction. Reducing chromatin compaction, which is known to soften the nucleus, enhances cell spread area and nuclear deformation but decreases F-actin accumulation and peripheral enrichment at the immune synapse. Conversely, enhancing chromatin compaction restricts spreading and deformation but promotes peripheral F-actin organization. This reveals a reciprocal relationship between chromatin compaction and cytoskeletal organization. We identify SUN proteins and myosin as key mediators through which chromatin compaction modulates actin morphology and cell shape, enabling T cells to adapt to antigen presenting surfaces of varying stiffness. These findings underscore the pivotal role of chromatin compaction in T cell activation, highlighting the mechanical interplay between the nucleus and cytoskeleton, and suggest novel insights into T cell mechano-responsiveness. [Media: see text] [Media: see text].
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, that mediate fusion at the cell surface, disrupt 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 mis-regulated, could contribute to the spread of neurodegenerative pathology.
During metaphase, chromosomes undergo oscillatory motion and exhibit distance-dependent coordinated movement with neighboring chromosomes within the spindle. However, the physical mechanism that gives rise to coordinated chromosome motion remains unresolved. Here, we combine quantitative live-cell imaging in PTK1 cells, targeted perturbations of spindle microtubules and chromatin condensation level, and minimal mechanical modeling to uncover the mechanical basis of chromosome coordination during metaphase. We show that chromosome oscillations are dampened by stabilizing microtubules or by reducing chromatin condensation, yet inter-chromosomal coordination measured by Pearson's correlation coefficient is preserved across all conditions. Consistently, simulations show that Pearson's correlation is insensitive to the mechanical parameters governing inter-chromosomal coupling. Together, these observations motivate the hypothesis that chromosome coordination is a mechanical signature of the physical spindle environment. To test this hypothesis, we develop a minimal mechanical model incorporating transient inter-chromosomal springs as a general representation of inter-chromosomal interactions, and show that they are sufficient to generate correlated chromosome motion. To quantify coordination in a manner that reflects the mechanical properties of the surrounding spindle environment, we used a microrheology-inspired analysis of time-lagged chromosome displacements applied to both experimental and simulated data, revealing that microtubules set the spatial range of coordination while chromatin condensation level modulates its strength. Together, our results support the hypothesis that coordinated chromosome motion is an emergent mechanical property of the mitotic spindle.
In recent years, substantial heterogeneity in lysosomes, in terms of their composition, function, and positioning, has come to be recognized. Despite this, there are gaps in knowledge of our understanding of the molecular basis of lysosome heterogeneity, especially in neurons. To this end, we used electron microscopy to define endolysosomal organelles of human iPSC-derived neurons at the ultrastructural level. Through this, we identify endolysosomal maturation defects within neuronal cell bodies of iNeurons lacking JNK-Interacting Protein 3 (JIP3), a lysosome adaptor previously known to primarily regulate axonal lysosome movement. Loss of JIP3 results in an expansion of immature lysosomes within neuronal soma, along with a concomitant decrease in mature lysosomes. JIP3 loss also leads to delayed trafficking of endocytic cargo through these compartments, implicating JIP3 in the regulation of endolysosomal maturation within neuronal cell bodies. Our studies highlight the utility of electron microscopy in understanding neuronal lysosomal heterogeneity. Additionally, given recent links between JIP3 and a neurodevelopmental disorder, our findings here could provide new insight into mechanisms underlying neurodevelopmental pathology.