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At least 19 recordsLinked to original sources

PubMed2025

Monitoring Influenza A Virus Entry Using Quantitative Fluorescence Microscopy.

Influenza A virus (IAV) is a major threat to global human health and is a topic of intense research. With the continuous problem of seasonal influenza and the threat of potential pandemics due to frequent emergence of new viral strains, development of new, broad-spectrum antivirals is an urgent priority. In antiviral development against influenza, the process of host cell entry of IAV is of particular interest as inhibiting the virus at the entry step should stop infection early on, blocking the downstream infection processes including viral replication and transcription. Therefore, a detailed understanding of the IAV entry processes is essential to illuminate virus-assisting host factors that can serve as potentially valuable targets for therapeutic interventions. To accelerate the identification of novel antivirals or host-directed targets that play essential role in IAV entry, quantitative assays that can be used to monitor the virus at sequential entry steps would be important for performing high-content genetic or inhibitor screens. In this chapter, we describe how IAV entry can be monitored at the sequential entry steps, spanning from the initial attachment of the virus particle to the cell surface to the transmission of the viral genome to the nucleus, by fluorescence microscopy. Further, we provide the methods to quantify the images acquired with high-content microscope for each of the major IAV entry steps. The fluorescence microscopy-based IAV entry assays and the image quantification methods described here can be used to boost our understanding of the virus-host cell interactions and can lead to the discovery of novel host-directed prophylactic or therapeutic interventions.

Humans↗

PubMed2025

Use of the D4H Probe to Track Sterols in Yeast.

Cholesterol is a fundamental component of cellular membranes, and its organization, distribution, and recycling are tightly regulated. Cholesterol can form, together with other lipids and proteins, membrane nanodomains, which play important roles in membrane trafficking, the spatiotemporal organization of signal transduction, or the modulation of plasma membrane transporters, among others. Not surprisingly then, the misregulation of cholesterol biosynthetic and transport pathways has been related to numerous diseases, including neurodegenerative and metabolic disorders. Here, we focus on the cholesterol-binding domain 4 (D4) of perfringolysin O (PFO, theta toxin) and its use as a probe to define the dynamics and subcellular localization of yeast sterols using time-lapse live-cell fluorescence microscopy. In combination with drugs that acutely interfere with sterol synthesis, such as terbinafine, the probe can also be used to monitor in real-time the extraction of sterols from specialized endoplasmic reticulum subdomains named ERSES (endoplasmic reticulum sterol exit sites) by the OSBP-related protein Osh2.

Saccharomyces cerevisiae↗

PubMed2026

Advanced High-Resolution and Super-Resolution Microscopy Techniques for Investigating Chromatin Structure and Epigenetics.

Fluorescence microscopy has emerged as an indispensable tool for investigating chromatin structure and epigenetic states. This technique not only enables super-resolution imaging to unveil detailed localizations and structures but also offers precise quantitative capabilities for comparing variations across multiple conditions. In this chapter, we present a comprehensive guide to immunofluorescence methods, detailing two primary fixation protocols. Additionally, we describe the application of super-resolution spinning disc microscopy, emphasizing its utility in detecting nuclear and epigenetic markers.

Chromatin↗

PubMed2025

Extracellular uncoating of bacteriophage MS2.

In the early stages of infection of its host, Escherichia coli, bacteriophage MS2 sheds its icosahedral protein capsid, after which the single-stranded genomic RNA (gRNA) and maturation protein enter the cell as a complex. Although the steps preceding uncoating, which include the binding of the Mat protein to the extracellular filament F-pilus, have been studied in detail, the uncoating step is not well understood. To study when and where uncoating happens, we image the infection process using fluorescence microscopy, separately labelling the MS2 capsid, its gRNA, and the cells. We do two types of experiments. In the first, we incubate the phage in a nonspecific intercalating dye, and we count the number of uncoated and intact phages before and after adding the labeled phages to cells. In the second, we examine the time course of infection by fixing unlabeled samples at different times after adding the phage, and then we label the MS2 gRNA using amplified fluorescence in situ hybridization. In both cases, we find that uncoating can occur anywhere on the F-pili, and that MS2 usually uncoats at a distance from the cell rather than at the cell surface. While these results do not rule out a current hypothesis that virus particles uncoat when the F-pilus retracts and brings them into contact with the cell body, they demonstrate an alternative, extracellular uncoating pathway. We discuss the possiblity that MS2 may have multiple uncoating pathways, and that the rate of each pathway could reflect a trade-off between different risk factors.

Levivirus↗

PubMed2026

Quantitative Fluorescence Imaging of Alphavirus Infection for Antiviral Screenings.

Fluorescence microscopy offers a highly sensitive and versatile approach for investigating alphavirus infection at the cellular level. By combining fluorescently labeled viruses with quantitative image analysis, this method enables detailed spatial and temporal characterization of infection dynamics, including the detection of subtle differences in replication kinetics and cell-to-cell spread. A central aim of this protocol is its application in antiviral screening assays. Image-based quantification of fluorescence intensity provides a robust and reproducible means to assess the efficacy of antiviral compounds, allowing early and sensitive detection of inhibitory effects in infected cells. This facilitates the identification of promising antiviral hits and supports the evaluation of dose-dependent responses. The approach is also well-suited for comparative studies of different alphavirus strains or mutants, as variations in replication behavior and dissemination patterns become readily apparent. Its flexibility, compatibility with multiple cell lines, and straightforward integration into automated imaging platforms makes the method scalable and suitable for high-throughput screening campaigns. Overall, this protocol advances the discovery and evaluation of antiviral strategies. Given that several alphaviruses cause significant human and veterinary diseases, lack approved antiviral therapies, and continue to expand geographically with emerging outbreaks, the identification of novel antivirals remains an urgent priority. Therefore, this fluorescence-based workflow represents a valuable and timely contribution to modern alphavirus research.

Antiviral Agents↗

PubMed2025

Zea mays Meiotic Spindle Ultrastructure Reveals Kinetochore-Microtubule Interface and Embedded Membrane Components.

UNLABELLED: Introduction: Spindles are microtubules-based machines whose primary function is to accurately segregate chromosomes in both mitotic and meiotic cell division. The structure of spindles is critical for their function; errors in morphology or attachment to chromosomes lead to aneuploidy, potentially resulting in disease, infertility, and lethality. Electron microscopy studies have yielded fine-detail spindle ultrastructures in many plant and animal species, but no studies have investigated the spindle of Zea mays, a critical crop, and cytogenetic model system. METHODS: Here we use electron tomography (ET), reconstruction, and modeling to obtain three-dimensional, nanometer-resolution of the Z. mays meiotic spindle. Structures such as microtubules, kinetochores, vesicles, membrane channels, and nuclear envelope were modeled through a partial spindle reconstruction, and confirmed using immunostaining and live fluorescence microscopy. RESULTS: ET revealed that maize spindles contain 8-18 kinetochore microtubules (kMTs) per kinetochore, which are approximately 776 nm in diameter and 316 nm in depth. Small ∼37 nm vesicles were identified, as well as larger (∼5 µm long, 800 nm wide) membrane structures with channels that allow spindle microtubules to pass through. These membrane channels stain positively for the ER-marker protein disulfide isomerase. Imaging of prophase meiotic cells revealed a cross-hatch microtubule arrangement in the perinuclear ring on the external surface of the nuclear envelope, which also contained type II nuclear grooves with transnuclear microtubules passing from the nucleus to the cytoplasm. CONCLUSIONS: Z. mays meiotic spindles are similar to animal counterparts with a comparable number of kMTs and pre-spindle transnuclear microtubules but also plant-specific features such as Golgi-derived vesicles to assist cell plate formation, internal ER membrane channels, and a perinuclear microtubule ring that aids spindle assembly. Maize kinetochores have an electron-diffuse ball in cup morphology that is comparable in size to Drosophila kinetochores and larger than mammalian kinetochores. .

Zea mays↗

PubMed2026

Mixing chromatin fibers with different nucleosome repeat lengths changes dynamics of chromatin phase separation.

The eukaryotic genome is organized into chromatin at multiple lengths and timescales. Liquid-liquid phase separation has recently emerged as a mechanism for the dynamic compartmentalization of chromatin. However, it remains unclear how cells can locally alter phase separation behavior to condense, decondense, and segregate specific regions of their genome. Selective interactions between chromatin fibers with different nucleosome repeat lengths (NRLs), as well as their incorporation into existing condensates composed of different NRL chromatin fibers, may provide a pathway for such processes. Using fluorescence microscopy, we investigated how these mechanisms influence the formation, coalescence, and maturation of chromatin condensates. Our results show distinct NRL-dependent mixing behaviors of chromatin before and after condensate formation. 167 and 197 NRL fibers, known to fold into compact fibers by strong nucleosome stacking interactions, formed amorphous condensates. In contrast, 172 and 202 NRL fibers, which only allow for weak stacking, formed spherical condensates. When NRLs were mixed, amorphous condensates exhibited localized clustering of identical NRLs. In spherical condensates, however, both NRLs were homogeneously distributed, with a varying NRL ratio per condensate. In addition, incorporation of 167 NRLs into preexisting 172 NRL condensates resulted in a multiphase structure where 167 NRL fibers formed an outer layer. These findings present an intrinsic link between DNA sequence, nucleosome positioning, local chromatin configuration and multiscale phase separation behavior. More broadly, they contribute to a deeper understanding of the dynamic methods of genome organization employed by eukaryotic organisms.

chromatin↗

PubMed2027

Lentiviral Transduction of Embryonic Stem Cells.

Lentiviral vectors provide an efficient and reliable method for stable gene knockdown in embryonic stem cells (ESCs) through RNA interference. Here, we describe a detailed protocol for lentiviral transduction of mouse ESCs using lentiviral shRNA expression vectors. The protocol encompasses lentiviral particle production in HEK-293T packaging cells, determination of viral titer, transduction of ESCs cultured under feeder-free conditions, and selection of stably transduced cells. Additionally, we describe methods for evaluating transduction efficiency using fluorescence microscopy and flow cytometry, as well as for assessing gene knockdown efficacy by quantitative real-time PCR (Q-RT-PCR). This protocol is suitable for functional genomic studies in pluripotent stem cells and can be adapted for other difficult-to-transfect cell types.

Lentivirus↗

PubMed2026

Antibacterial activity and mechanistic insights of Lucilia illustris antimicrobial peptide Cecropin A2 against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) poses a serious public health threat due to multidrug resistance and biofilm formation. This study investigated the antibacterial mechanisms of the antimicrobial peptide, Cecropin A2 (CA2), against P. aeruginosa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CA2 against P. aeruginosa ATCC 9027 (PA ATCC 9027) were determined by broth microdilution. Antibacterial activity was evaluated using growth curves and time-kill assays. The mechanism was explored by assessing membrane integrity (outer/inner membrane permeability, SEM, and fluorescence microscopy), and intracellular responses (ATP, SDH activity, and ROS). Biofilm effects were assessed by crystal violet staining (biomass) and viable cell counting (biofilm-embedded bacteria). The MIC and MBC of CA2 against PA ATCC 9027 were 37.34 μM and 74.68 μM, respectively. CA2 exhibited moderate antibacterial activity against PA ATCC 9027. Scanning electron microscopy (SEM) revealed marked morphological damage after treatment. CA2 affected intracellular metabolism, potentially interacted with genomic DNA, and reduced biofilm biomass. Cecropin A2 exhibits concentration-dependent in vitro antibacterial activity against P. aeruginosa ATCC 9027, providing mechanistic insights and a theoretical basis for the development of alternative antimicrobial strategies.

Antimicrobial activity↗

PubMed2026

A set of genetic tools for use in Clostridioides difficile and related species.

The Clostridia are a phylogenetically diverse group of anaerobic, spore-forming bacteria that include species of medical, veterinary and industrial importance. The last two decades have seen major advances in our understanding of Clostridial biology despite the difficulties of anaerobic microbiology and the challenges associated with limited genetic tools. Effort has largely focused on the human pathogen Clostridioides difficile, but many of the methods developed have also proven useful in other species. Here, we present a collection of new genetic tools, including an array of promoters of varying strength, that we have characterized in C. difficile, the food spoilage bacterium Clostridium sporogenes and industrially important Clostridium saccharoperbutylacetonicum. We also present a set of modular plasmids that allow expression of proteins with a variety of tags, including for protein purification and fluorescence microscopy and a method for genetic barcoding of C. difficile to facilitate competitive index experiments. We make these tools available in the hope that they will prove useful to the community in support of our growing understanding of these important bacteria.

Clostridioides difficile↗

PubMed2025

Subcellular proteomics of the protist Paradiplonema papillatum reveals the digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans.

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate-degrading enzymes (CDZymes). Our predictions suggest that these CDZymes are exposed to the extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

Peroxisomes↗

PubMed2025

Modularization of the type II secretion gene cluster from Xanthomonas euvesicatoria facilitates the identification of a structurally conserved XpsCLM assembly platform complex.

Many bacterial pathogens depend on a type II secretion (T2S) system to secrete virulence factors from the periplasm into the extracellular milieu. T2S systems consist of an outer membrane secretin channel, a periplasmic pseudopilus and an inner membrane-associated assembly platform including a cytoplasmic ATPase. The components of T2S systems are often conserved in different bacterial species, however, the architecture of the assembly platform is largely unknown. Here, we analysed predicted assembly platform components of the Xps-T2S system from the plant-pathogenic bacterium Xanthomonas euvesicatoria. To facilitate these studies, we generated a modular xps-T2S gene cluster by Golden Gate assembly of single promoter and gene fragments. The modular design allowed the efficient deletion and replacement of T2S genes and the insertion of reporter fusions. Mutant approaches as well as interaction and crosslinking studies showed that the predicted assembly platform components XpsC, XpsL and XpsM form a trimeric complex which is essential for T2S and associates with the cytoplasmic ATPase XpsE and the secretin XpsD. Structural modeling revealed a similar trimeric architecture of XpsCLM homologs from Pseudomonas, Vibrio and Klebsiella species, despite overall low amino acid sequence similarities. In X. euvesicatoria, crosslinking and fluorescence microscopy studies showed that the formation of the XpsCLM complex is independent of the secretin and vice versa, suggesting that the assembly of the T2S system is a dynamic process which involves the association of preformed subcomplexes.

Xanthomonas↗

PubMed2025

Functional characterization of DPP4 and FcRn as receptor and coreceptor for classical human astroviruses in Caco-2 cells.

Classical human astroviruses (HAstV) are a global cause of viral gastroenteritis, particularly in children and immunocompromised individuals. Despite their clinical significance, the biology of HAstV remains poorly understood. In particular, the identification of cellular receptors and coreceptors has been elusive. Recent studies have identified the human neonatal Fc receptor (FcRn) as a functional receptor and dipeptidyl peptidase IV (DPP4) as an entry factor for HAstV. However, the precise roles of FcRn and DPP4 during HAstV infection are unknown. To learn about their function, we used FcRn-knockout (KO), DPP4-KO, and FcRn/DPP4 double-KO Caco-2 cells generated via CRISPR/Cas9. Our results showed that DPP4 serves as the receptor for classical HAstV. In contrast, infectious virus assays and confocal fluorescence microscopy revealed that FcRn acts as a coreceptor, facilitating viral internalization and the release of the RNA genome. The half-time for HAstV-1 genome uncoating was delayed threefold in FcRn-KO Caco-2 cells compared to WT cells. Additionally, the characterization of HAstV-8 variants with reduced FcRn binding capacity allowed the identification of two amino acids in the viral capsid spike protein, D471 and N512, critical for the spike-FcRn interaction. These amino acid residues are part of the epitope footprint of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV previously mapped by X-ray crystallography. Further experiments using virus infectivity and attachment assays, along with Nt-MAbs targeting HAstV-1, suggest that the binding sites for FcRn and DPP4 are spatially proximal on the viral spike, defining a functional domain for cell infection. Notably, the infectivity of the divergent HAstV-VA1 was independent of these two proteins, highlighting the receptor variability across HAstV clades. These findings provide new insights into the mechanism of HAstV infection, offering relevant implications for the development of antiviral therapies and vaccines targeting this significant human pathogen.

Humans↗

PubMed2015

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

Alleles↗

PubMed2002

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression.

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress-induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear-induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow-induced stimulation of nuclear factor kappaB (NF-kappaB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM-1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.

Animals↗

PubMed2025

3D STED Imaging of Isolated Arabidopsis thaliana Nuclei.

Microscopy imaging of chromatin offers valuable insights into its spatial organization in the nucleus, a novel epigenetic dimension influencing the genome's functions. Particularly, visualization at the nanoscale in single cells is uniquely complementary to molecular profiling methods averaging chromatin configuration and composition over thousands of cells. How are chromatin and chromosomal domains distributed in relation to gene expression? How variable are these configurations? How do chromatin domains evolve in structure, composition, and distribution during cellular differentiation or cellular responses to environmental stimuli? Super-resolution microscopy techniques, like stimulated emission depletion (STED), are key in answering such questions. However, such imaging techniques are not often used in the field of plant cell biology compared to mammalian counterparts, which has greatly advanced our understanding of the 3D principles in genome organization. In an effort to bridge this gap, we provide a clear guide for isolating, embedding, immunostaining, and STED imaging intact leaf nuclei from Arabidopsis thaliana in 3D.

Arabidopsis↗

PubMed2002

A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system.

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.

Amino Acid Sequence↗

PubMed2025

FRET-FLIM for the Study of Protein-Protein Interactions Underpinning Mitosis Checkpoints.

Cell division is a key cellular process that ensures the continuation of life on Earth. In order to protect the genetic integrity of organisms, cell division must happen accurately, ensuring each daughter cell receives a complete copy of the original genome. The accuracy of this process is, in part, preserved by various cell cycle checkpoints. These checkpoints rely on the physical interactions of their components to ensure proper function. The spindle assembly checkpoint (SAC), for example, produces an inhibitory complex of BUBR1-BUB3 and MAD2 bound to CDC20. Many of these cell cycle checkpoint components have been identified in plants, but it has not yet been established whether plants have a mitotic checkpoint architecture that is similar to mammalian cells. To understand the function of plant cell cycle homologues, it is imperative to characterize their interactions in vivo. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy), is a rapidly expanding technique that can be used to rapidly and simply characterize protein-protein interactions.

Fluorescence Resonance Energy Transfer↗