Live-cell Fluorescence Microscopy Research Articles

Bromodomains regulate dynamic targeting of the PBAF chromatin-remodeling complex to chromatin hubs

Chromatin remodelers actively target arrays of acetylated nucleosomes at select enhancers and promoters to facilitate or shut down the repeated recruitment of RNA polymerase II during transcriptional bursting. It is poorly understood how chromatin remodelers such as PBAF dynamically target different chromatin states inside a live cell. Our live-cell single-molecule fluorescence microscopy study reveals chromatin hubs throughout the nucleus where PBAF rapidly cycles on and off the genome. Deletion of PBAF’s bromodomains impairs targeting and stable engagement of chromatin in hubs. Dual color imaging reveals that PBAF targets both euchromatic and heterochromatic hubs with distinct genome-binding kinetic profiles that mimic chromatin stability. Removal of PBAF’s bromodomains stabilizes H3.3 binding within chromatin, indicating that bromodomains may play a direct role in remodeling of the nucleosome. Our data suggests that PBAF’s dynamic bromodomain-mediated engagement of a nucleosome may reflect the chromatin-remodeling potential of differentially bound chromatin states.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y1 Receptor.

The recent crystallization of the neuropeptide Y Y1 receptor (Y1R) in complex with the argininamide-type Y1R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design of nonpeptidic Y1R ligands. We designed novel fluorescent probes showing excellent Y1R selectivity and, in contrast to previously described fluorescent Y1R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y1R binding affinities (pKi values of 9.36-9.95) and proved to be Y1R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry- and fluorescence anisotropy-based Y1R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.

Modeling simultaneous two-wavelength axial ratiometry imaging of clathrin mediated endocytosis

Clathrin-mediated endocytosis (CME) facilitates the internalization of extracellular cargoes. However, how clathrin-coated vesicles (CCVs) form remains unclear due to the limited resolution of live-cell fluorescence microscopy and the need for sample fixation in electron microscopy. To bridge this gap, our lab developed Simultaneous Two-wavelength Axial Ratiometry (STAR) that leverages the wavelength-dependent properties of total internal reflection fluorescence microscopy. Dual-tagging a protein of interest with two spectrally separated fluorophores allows STAR to measure the intensity ratio and retrieve the z-position of the protein.

Live Fluorescence Imaging of Chromosome Segregation in Cultured Cells.

Live-cell fluorescence microscopy is an effective tool for characterizing aberrant mitotic phenotypes resulting from exposure to chemical inhibitors and after RNA interference-mediated or CRISPR knockout-mediated depletion of protein targets. Live imaging of cultured cells during mitotic progression presents challenges in maintaining optimal health of cells while achieving the temporal and spatial resolution to accomplish the goals of the study. Herein are strategies to monitor and analyze mammalian cell mitosis utilizing either a wide field or a light sheet, inverted, fluorescence microscope.

Development of vibration mechanical stimuli loading device for live cell fluorescence microscopy

Development of vibration mechanical stimuli loading device for live cell fluorescence microscopy

Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells.

Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

Imaging of the Cytoskeleton Using Live and Fixed Drosophila Tissue Culture Cells.

In recent years, the convergence of multiple technologies and experimental approaches has led to the expanded use of cultured Drosophila cells as a model system. Their ease of culture and maintenance, susceptibility to RNA interference, and imaging characteristics have led to extensive use in both traditional experimental approaches and high-throughput RNAi screens. Here we describe Drosophila S2 cell culture and preparation for live-cell and fixed-cell fluorescence microscopy and scanning electron microscopy.

An In Vitro Model System to Test Mechano-Microbiological Interactions Between Bacteria and Host Cells.

The aim of this chapter is to present an innovative technique to visualize changes of the F-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust F-actin accumulation in individual cells at the sites where pulling forces were applied. Using the magnetic tweezers, we were able to mimic the local response of the F-actin cytoskeleton to bacteria-generated forces. In this chapter, we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.

The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

How to control fluorescent labeling of metal oxide nanoparticles for artefact-free live cell microscopy

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles—the largest subpopulation of nanoparticles by industrial production and applications—and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles’ surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials’ mechanism of action and will foster widespread and safe use of new nanomaterials.

Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy.

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or a spontaneously blinking dye for single-molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

Antimicrobial Peptides: Delivery by Dendritic Mesoporous Silica Nanoparticles Enhances the Antimicrobial Activity of a Napsin‐Derived Peptide Against Intracellular Mycobacterium tuberculosis (Adv. Healthcare Mater. 14/2021)

In article number 2100453 by Steffen Stenger, Mika Lindén, and co-workers, the activity of the antimicrobial peptide NapFab against intracellular Mycobacterium tuberculosis (Mtb) is significantly increased by loading onto dendritic mesoporous silica nanoparticles (DMSN). This enhanced activity is based on a 30-fold increased, DMSN-mediated peptide uptake. Livecell fluorescence microscopy and electron microscopy reveal fast DMSN uptake in endosomal vesicles, intracellular degradation of the carrier, and a continuous peptide release.

Abstract 1045: Combined targeting of actin/tropomyosin and microtubules underlies a potential treatment strategy of epithelial ovarian cancer with cell-cycle dependent synergy

Abstract Anti-microtubule agents are widely used to treat ovarian cancers in the first line and recurrent setting either in combination with platinum or as single agents. However, the majority of patients will experience a recurrence, and most will die with drug resistant disease. We investigated co-targeting the actin cytoskeleton in combination with anti-microtubule agents to increase efficacy of treatment in epithelial ovarian cancers and potentially overcome resistance mechanisms. We examined the presence of actin/tropomyosin 3.1 (Tpm3.1) filaments in a large cohort of clinical specimens from patients with epithelial ovarian cancer of all histotypes using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound (ATM) with both vinorelbine and paclitaxel were evaluated in three ovarian cancer cell lines using cell viability and apoptosis assays. The mechanisms of synergy of both combinations were established using live-cell imaging, fluorescent microscopy, and pathway analysis. We found that Tpm3.1 is abundant and overexpressed in 97% of ovarian cancers examined (558 of 577) representing all histotypes of epithelial ovarian cancer. High levels of Tpm3.1 were also present in all sites sampled and similar at primary diagnosis and at recurrence. ATM displayed both single agent activity as well as synergy with both anti-microtubule drugs to reduce cell viability in all ovarian cancer cell lines tested, including one with platinum resistance. Only vinorelbine, however, synergised with ATM in the induction of apoptosis. Vinorelbine-induced mitotic arrest was significantly prolonged by ATM with elevated activity of the spindle assembly checkpoint, leading to almost one third of total cells dying in mitosis. In contrast, ATM showed minor impact on paclitaxel-induced mitotic defects. Both combinations resulted in a substantial increase in cells arrested in the subsequent G1 phase with a large decrease of both cyclin D1 and E1 as compared to single agents. Upregulation of p21Cip and p27Kip were associated with both combinations. In summary, targeting Tpm3.1-associated actin filaments in combination with anti-microtubule drugs is a promising treatment strategy that should be tested in clinical trials and is potentially applicable to all histotypes of ovarian cancer. Citation Format: Xing Xu, Yao Wang, Nicole Bryce, Katrina Tang, Nicola S. Meagher, Eun Young Kang, Linda E. Kelemen, Martin Köbel, Susan J. Ramus, Michael Friedlander, Caroline E. Ford, Edna C. Hardeman, Peter W. Gunning. Combined targeting of actin/tropomyosin and microtubules underlies a potential treatment strategy of epithelial ovarian cancer with cell-cycle dependent synergy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1045.

Gland cell responses to feeding in Drosera capensis, a carnivorous plant

Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Nonparametric detection of changes over time in image data from fluorescence microscopy of living cells

AbstractThe question whether structural changes in time‐resolved images are of statistical significance or merely emerge from random noise is of great relevance in many practical applications such as live cell fluorescence microscopy, where intracellular diffusion processes are investigated. Using bootstrap‐methods, we construct nonparametric confidence bands for time‐resolved images from fluorescence microscopy and use these to detect and visualize temporal changes between individual frames in imaging of living cells. We model the images frames as two‐dimensional fields of Poisson random variables and provide a strong approximation result for independent and standardized but not necessarily identically distributed Poisson random variables. The latter result is used to derive a limit result for the maximal difference between the reconstructed and the true image. This provides the theoretical foundation of our method. We apply regularization techniques to cope with the ill‐posedness of the convolution problem induced by the imaging system. Our approach provides a criterion to assess time‐resolved small scale structural changes, for example, in the nanometer range. It can also be adopted for use in other imaging systems. Moreover, a data‐driven selection method for the regularization parameter based on statistical multiscale methods is discussed.

Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.

Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging

Lectins are involved in a wide range of carbohydrate mediated recognition processes. Therefore, the availability of highly performant fluorescent tools tailored for lectin targeting and able to efficiently track events related to such key targets is in high demand. We report here on the synthesis of the glyco-BODIPYs 1 and 2, based on the efficient combination of a Heck-like cross coupling and a Knoevenagel condensation, which revealed efficient in addressing lectins. In particular, glyco-BODIPY 1 has two glycosidase stable C-mannose residues, which act as DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) targeting modules. By using live-cell fluorescence microscopy, we proved that BODIPY-mannose 1 was efficiently taken up by immune cells expressing DC-SIGN receptors. Super-resolution stimulated emission depletion (STED) microscopy further revealed that the internalized 1 localized in membranes of endosomes, proving that 1 is a reliable tool also in STED applications. Of note, glyco-BODIPY 1 contains an aryl-azido group, which allows further functionalization of the glycoprobe with bioactive molecules, thus paving the way for the use of 1 for tracking lectin-mediated cell internalization in diverse biological settings.