Live-cell Microscopy Research Articles

Antagonistic Effects of Actin-Specific Toxins on Salmonella Typhimurium Invasion into Mammalian Cells

Competition between bacterial species is a major factor shaping microbial communities. It is possible but remains largely unexplored that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by actin-specific toxins, namely, Vibrio cholerae MARTX actin crosslinking (ACD) and Rho GTPase inactivation (RID) domains, Photorhabdus luminescens TccC3, and Salmonella’s own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin-binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays and by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. These results suggest that bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria.

Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy

We have developed a method combining microinjection and automated fluorescence microscopy to continuously assess the degradation rate, subcellular localization and intracellular concentration of protein analytes at the single-cell level. Cells are unperturbed and grown in unaltered environmental conditions and show high viability. The injection of analytes at defined ratios and concentrations allows for a clearly defined starting point of degradation, without the entanglement of biosynthesis/uptake, often encountered in existing methods. The possibility to evaluate, add, or remove post-translational modifications prior to injection represents a powerful tool to assess minute protein degradation rate changes with high precision and allowed us to determine the absolute degradation rates caused by N-degron pathway engagement, with a focus on the role of acetylation. The low degradation rate of eGFP was found to be caused by inefficient N-terminal proteasomal unfolding. We moreover quantified the surprisingly strong influences of commonly used peptide tags and detected high variation between fluorescent proteins with regard to both protein degradation and subcellular localization. Furthermore, we have validated the use of chemically coupled dyes as robust reporters for protein degradation, and elucidated the significance of their membrane-permeability, thereby extending the applicability of our method to any protein of interest.

Single-cell analysis of osmoregulation reveals heterogeneity of aquaporin 4 functionality in human astrocytes.

The water channel aquaporin 4 (AQP4) contributes to water flow and waste removal across the blood-brain barrier and its levels, organization and localization are perturbed in various neurological diseases, including Alzheimer's Disease. This renders AQP4 a potentially valuable therapeutic target. However, most functional assays aimed at identifying modulators of AQP4 function are performed with primary rodent cells and do not consider inter-cellular variations in AQP4 abundance and presentation. To address this, we have established and applied a robust live cell microscopy assay that captures the contribution of AQP4 in the osmotically driven (de-)quenching of the vital dye Calcein-AM with single-cell resolution. Using human astrocytoma cells, we found that performing measurements on cellular regions instead of whole fields of view yielded a more sensitive readout of the osmotic response, which correlated with AQP4 abundance. Stable co-expression of the two major AQP4 isoforms, but not of the individual isoforms, provoked a faster adaptation to osmotic changes, while siRNA-mediated knockdown of AQP4 had the opposite effect. Post-hoc correlation with the canonical membrane marker CD44 revealed that the speed of the osmotic response scaled with AQP4 membrane enrichment. Coating the substrate with laminin promoted AQP4 membrane enrichment, while cell confinement with fixed-size micropatterns further increased the speed of osmoregulation, underscoring the influence of extracellular factors. The osmotic response of primary fetal astrocytes and human iPSC-derived astrocyte models was comparable to AQP4-deficient astrocytoma cells, in line with their low AQP4 levels and indicative of their immature state. In conclusion, a correlative single-cell approach based on the quantification of Calcein-AM quenching capacity, AQP4 abundance and AQP4 membrane enrichment, allows resolving osmoregulation in a more sensitive manner and reveals heterogeneity between and within human astrocyte (-like) cultures, which could prove crucial for future screens aimed at identifying AQP4 modulators.

Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.

BackgroundBabesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.MethodsThe endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).ResultsIn this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.ConclusionsThis study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.Graphical

A programmable platform for probing cell migration and proliferation.

The advent of advanced robotic platforms and workflow automation tools has revolutionized the landscape of biological research, offering unprecedented levels of precision, reproducibility, and versatility in experimental design. In this work, we present an automated and modular workflow for exploring cell behavior in two-dimensional culture systems. By integrating the BioAssemblyBot® (BAB) robotic platform and the BioApps™ workflow automater with live-cell fluorescence microscopy, our workflow facilitates execution and analysis of in vitro migration and proliferation assays. Robotic assistance and automation allow for the precise and reproducible creation of highly customizable cell-free zones (CFZs), or wounds, in cell monolayers and "hands-free," schedulable integration with real-time monitoring systems for cellular dynamics. CFZs are designed as computer-aided design models and recreated in confluent cell layers by the BAB 3D-Bioprinting tool. The dynamics of migration and proliferation are evaluated in individual cells using live-cell fluorescence microscopy and an in-house pipeline for image processing and single-cell tracking. Our robotics-assisted approach outperforms manual scratch assays with enhanced reproducibility, adaptability, and precision. The incorporation of automation further facilitates increased flexibility in wound geometry and allows for many experimental conditions to be analyzed in parallel. Unlike traditional cell migration assays, our workflow offers an adjustable platform that can be tailored to a wide range of applications with high-throughput capability. The key features of this system, including its scalability, versatility, and the ability to maintain a high degree of experimental control, position it as a valuable tool for researchers across various disciplines.

On the axonal transport of lipid nanoparticles in primary hippocampal neurons

Axonal transport is a crucial process in healthy neurons as it supports the intra-cellular movement of nutrients, endogenous substances, and vesicles regulating a broad set of biological functions. Notably, this physiological mechanism is efficiently exploited by a variety of viruses to infect multiple cells within the central nervous system and, thus, it has been proposed as a strategy to enhance the brain penetrance of macromolecules and nanoparticles. In this work, the retrograde and anterograde transport of lipid nanoparticles (LNP) is systematically analyzed in primary hippocampal neurons cultured in compartmentalized microfluidic chips, where neurites are left to grow within 150 μm-long channels connecting the somal and synaptic compartments. After characterizing the physico-chemical properties, toxicological profile, and cell internalization efficiency, the axonal trafficking of fluorescently labeled LNP was monitored over time via live-cell microscopy. Both naïve LNP and apolipoprotein E-coated LNP (ApoE-LNP) were considered under two different experimental configurations, with the LNP being either added to the somal or the synaptic compartment for anterograde or retrograde transport analyses, respectively. ApoE-LNP only were very efficiently uptaken by neurons and rapidly relocated in a perinuclear position. Also, ApoE-LNP incubated in the somal compartment did not translocate along the neurites (null anterograde transport), whereas ApoE-LNP added to the synaptic compartment were detected near the soma already at 30 min post incubation demonstrating retrograde transport velocities up to ∼ 160 nm/s. This preliminary study suggests that ApoE-LNP could be efficiently used to rapidly transport a variety of therapeutic and imaging cargos from the synaptic cleft to the somal compartment.

Molecular and microbiological methods for the identification of nonreplicating Mycobacterium tuberculosis.

Chronic tuberculosis (TB) disease, which requires months-long chemotherapy with multiple antibiotics, is defined by diverse pathological manifestations and bacterial phenotypes. Targeting drug-tolerant bacteria in the host is critical to achieving a faster and durable cure for TB. In order to facilitate this field of research, we need to consider the physiology of persistent MTB during infection, which is often associated with the nonreplicating (NR) state. However, the traditional approach to quantifying bacterial burden through colony enumeration alone only informs on the abundance of live bacilli at the time of sampling, and provides an incomplete picture of the replicative state of the pathogen and the extent to which bacterial replication is balanced by ongoing cell death. Modern approaches to profiling bacterial replication status provide a better understanding of inter- and intra-population dynamics under different culture conditions and in distinct host microenvironments. While some methods use molecular markers of DNA replication and cell division, other approaches take advantage of advances in the field of microfluidics and live-cell microscopy. Considerable effort has been made over the past few decades to develop preclinical in vivo models of TB infection and some are recognized for more closely recapitulating clinical disease pathology than others. Unique lesion compartments presenting different environmental conditions produce significant heterogeneity between Mycobacterium tuberculosis populations within the host. While cellular lesion compartments appear to be more permissive of ongoing bacterial replication, caseous foci are associated with the maintenance of M. tuberculosis in a state of static equilibrium. The accurate identification of nonreplicators and where they hide within the host have significant implications for the way novel chemotherapeutic agents and regimens are designed for persistent infections.

5119 Structural basis for glucocorticoid receptor multimerization

Abstract Disclosure: E. Estébanez-Perpiñá: None. Abstract ENDO-2024 - BostonStructural basis for glucocorticoid receptor multimerization Andrea Alegre-Martí[1]*, Alba Jimenez-Panizo2*, Agustina Lafuente3, Montserrat Abella[1], Thomas A Johnson2, Paloma Pérez4, Juan Fernández-Recio5, Diego M. Presman3, Gordon L. Hager2, Pablo Fuentes-Prior[1], Eva Estébanez-Perpiñá[1] [1]Department of Biochemistry and Molecular Biomedicine, Institute of Biomedicine of the University of Barcelona (IBUB), Faculty of Biology, University of Barcelona (UB), 08028 Barcelona, Spain.2National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5055, USA.3IFIBYNE, UBA-CONICET, Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, C1428EGA, Argentina.4Instituto de Biomedicina de Valencia (IBV)-CSIC, 46010, Valencia, Spain.5Instituto de Ciencias de la Vid y del Vino (ICVV), CSIC - Universidad de La Rioja - Gobierno de La Rioja, 26007 Logroño, Spain. Abstract: The glucocorticoid receptor (GR) is a ubiquitously e2xpressed ligand-regulated transcription factor essential for life and one of the most targeted proteins in drug discovery due to its powerful anti-inflammatory actions. The functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity in cells, remains disputed. Here we present a new crystal structure of agonist-bound ancient GR-LBD in a large cell, along with a thorough analysis of previous structural work. The building block of the current structure is a homodimer we previously identified in GR-LBD crystals and its biological relevance has been verified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in live cells. Several mutually exclusive multimeric assemblies of this dimer in the crystal highlight the versatility of GR-LBD for self-association and reveal implications for the conformation of the active full-length receptor. Our results underscore the relevance of non-canonical dimerization modes for GR-LBD, especially of contacts made by key residues such as Tyr545, Pro637 and Asp641. Of note, a non-conservative mutation of the latter, p.Asp641Val, causes Chrousos syndrome in humans. Understanding relevant quaternary assemblies of the GR is pivotal not only to understand and predict the therapeutic outcome of major blockbuster drugs but also to lessen their deleterious side effects and open new avenues for drug design. Presentation: 6/3/2024

Targeted Photoactivatable Green-Emitting BODIPY Based on Directed Photooxidation-Induced Activation and its Application to Live Dynamic Super-Resolution Microscopy.

Photoactivatable fluorescent probes are valuable tools in bioimaging for tracking cells down to single molecules and for single molecule localization microscopy. For the latter application, green emitting dyes are in demand. We herein developed an efficient green-emitting photoactivatable furanyl-BODIPY (PFB) and we established a new mechanism of photoactivation called Directed Photooxidation Induced Activation (DPIA) where the furan is photo-oxidized in a directed manner by the singlet oxygen produced by the probe. The efficient photoconverter (93-fold fluorescence enhancement at 510 nm, 49% yield conversion) is functionalizable and allowed targeting of several subcellular structures and organelles, which were photoactivated in live cells. Finally, we demonstrated the potential of PFB in super-resolution imaging by performing PhotoActivated Localization Microscopy (PALM) in live cells.

Self-inspired learning for denoising live-cell super-resolution microscopy.

Every collected photon is precious in live-cell super-resolution (SR) microscopy. Here, we describe a data-efficient, deep learning-based denoising solution to improve diverse SR imaging modalities. The method, SN2N, is a Self-inspired Noise2Noise module with self-supervised data generation and self-constrained learning process. SN2N is fully competitive with supervised learning methods and circumvents the need for large training set and clean ground truth, requiring only a single noisy frame for training. We show that SN2N improves photon efficiency by one-to-two orders of magnitude and is compatible with multiple imaging modalities for volumetric, multicolor, time-lapse SR microscopy. We further integrated SN2N into different SR reconstruction algorithms to effectively mitigate image artifacts. We anticipate SN2N will enable improved live-SR imaging and inspire further advances.

Long-range repulsion between chromosomes in mammalian oocyte spindles.

During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them.

A temperature-inducible protein module for control of mammalian cell fate.

Inducible protein switches allow on-demand control of proteins in response to inputs including chemicals or light. However, these inputs either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. The original Melt variant switched states between 28-32°C, and state changes could be observed within minutes of temperature change. Melt was highly modular, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements, and cell death, all through straightforward end-to-end fusions. Melt was also highly tunable, giving rise to a library of variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer, demonstrating its potential for use in animals. Thus Melt represents a versatile thermogenetic module for straightforward, non-invasive, spatiotemporally-defined control of mammalian cells with broad potential for biotechnology and biomedicine.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

Tip extension and simultaneous multiple fission in a filamentous bacterium

Organisms display an immense variety of shapes, sizes, and reproductive strategies. At microscopic scales, bacterial cell morphology and growth dynamics are adaptive traits that influence the spatial organization of microbial communities. In one such community-the human dental plaque biofilm-a network of filamentous Corynebacterium matruchotii cells forms the core of bacterial consortia known as hedgehogs, but the processes that generate these structures are unclear. Here, using live-cell time-lapse microscopy and fluorescent D-amino acids to track peptidoglycan biosynthesis, we report an extraordinary example of simultaneous multiple division within the domain Bacteria. We show that C. matruchotii cells elongate at one pole through tip extension, similar to the growth strategy of soil-dwelling Streptomyces bacteria. Filaments elongate rapidly, at rates more than five times greater than other closely related bacterial species. Following elongation, many septa form simultaneously, and each cell divides into 3 to 14 daughter cells, depending on the length of the mother filament. The daughter cells then nucleate outgrowth of new thinner vegetative filaments, generating the classic "whip handle" morphology of this taxon. Our results expand the known diversity of bacterial cell cycles and help explain how this filamentous bacterium can compete for space, access nutrients, and form important interspecies interactions within dental plaque.

Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression.

This study focuses on the role of lysosomal trafficking in prostate cancer, given the essential role of lysosomes in cellular homoeostasis. Lysosomal motility was evaluated using confocal laser scanning microscopy of LAMP-1-transfected prostate cells and spot-tracking analysis. Expression of lysosomal trafficking machinery was evaluated in patient cohort databases and through immunohistochemistry on tumour samples. The roles of vesicular trafficking machinery were evaluated through over-expression and siRNA. The effects of R1881 treatment on lysosome vesicular trafficking was evaluated by RNA sequencing, protein quantification and fixed- and live-cell microscopy. Altered regulation of lysosomal trafficking genes/proteins was observed in prostate cancer tissue, with significant correlations for co-expression of vesicular trafficking machinery in Gleason patterns. The expression of trafficking machinery was associated with poorer patient outcomes. R1881 treatment induced changes in lysosomal distribution, number, and expression of lysosomal vesicular trafficking machinery in hormone-sensitive prostate cancer cells. Manipulation of genes involved in lysosomal trafficking events induced changes in lysosome positioning and cell phenotype, as well as differential effects on cell migration, in non-malignant and prostate cancer cells. These findings provide novel insights into the altered regulation and functional impact of lysosomal vesicular trafficking in prostate cancer pathogenesis.

Chiral Fluorescent Antifungal Azole Probes Detect Resistance, Uptake Dynamics, and Subcellular Distribution in Candida Species.

Azoles are essential for fungal infection treatment, yet the increasing resistance highlights the need for innovative diagnostic tools and strategies to revitalize this class of antifungals. We developed two enantiomers of a fluorescent antifungal azole probe (1 S and 1 R ), analyzing 60 Candida strains via live-cell microscopy. A database of azole distribution images in strains of Candida albicans, Candida glabrata, and Candida parapsilosis, among the most important pathogenic Candida species, was established and analyzed. This analysis revealed distinct populations of yeast cells based on the correlation between fluorescent probe uptake and cell diameter. Varied uptake levels and subcellular distribution patterns were observed in C. albicans, C. glabrata, and C. parapsilosis, with the latter displaying increased localization to lipid droplets. Comparison of the more potent fluorescent antifungal azole probe enantiomer 1 S with the moderately potent enantiomer 1 R highlighted time-dependent differences in the uptake profiles. The former displayed a marked elevation in uptake after approximately 150 min, indicating the time required for significant cell permeabilization to occur and its association with the azole's antifungal activity potency. Divergent uptake levels between susceptible and high efflux-based azole-resistant strains were detected, offering a rapid diagnostic approach for identifying azole resistance. This study highlights unique insights achievable through fluorescent antifungal azole probes, unraveling the complexities of azole resistance, subcellular dynamics, and uptake within fungal pathogens.

Kinetic Landscape of Single Virus-like Particles Highlights the Efficacy of SARS-CoV-2 Internalization.

The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.

Downregulation of Fidgetin-Like 2 Increases Microglial Function: The Relationship Between Microtubules, Morphology, and Activity.

The microtubule cytoskeleton regulates microglial morphology, motility, and effector functions. The microtubule-severing enzyme, fidgetin-like 2 (FL2), negatively regulates cell motility and nerve regeneration, making it a promising therapeutic target for central nervous system injury. Microglia perform important functions in response to inflammation and injury, but how FL2 affects microglia is unclear. In this study, we investigated the role of FL2 in microglial morphology and injury responses in vitro. We first determined that the pro-inflammatory stimulus, lipopolysaccharide (LPS), induced a dose- and time-dependent reduction in FL2 expression associated with reduced microglial ramification. We then administered nanoparticle-encapuslated FL2 siRNA to knockdown FL2 and assess microglial functions compared to negative control siRNA and vehicle controls. Time-lapse live-cell microscopy showed that FL2 knockdown increased the velocity of microglial motility. After incubation with fluorescently labeled IgG-opsonized beads, FL2 knockdown increased phagocytosis. Microglia were exposed to low-dose LPS after nanoparticle treatment to model injury-induced cytokine secretion. FL2 knockdown enhanced LPS-induced cytokine secretion of IL-1α, IL-1β, and TNFα. These results identify FL2 as a regulator of microglial morphology and suggest that FL2 can be targeted to increase or accelerate microglial injury responses.

Live-cell analysis of IMPDH protein levels during yeast colony growth provides insights into the regulation of GTP synthesis.

This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability.