Time-lapse Fluorescence Microscopy Research Articles

Competing Interactions of Fatty Acids and Monoglycerides Trigger Synergistic Phospholipid Membrane Remodeling.

Using quartz crystal microbalance-dissipation and time-lapse fluorescence microscopy, we demonstrate that adding mixtures of lauric acid (LA) and glycerol monolaurate (GML), two of the most biologically active antimicrobial fatty acids and monoglycerides, to a supported lipid bilayer triggers concurrent tubule and bud formation, which unexpectedly results in synergistic phospholipid membrane remodeling that far exceeds the effects of GML or LA alone. Together, GML and LA drive pearling instability, dynamic transformation of buds into tubules and vice versa, and extensive membrane lysis. The most pronounced effects occurred with equimolar concentrations of GML and LA, highlighting that synergistic membrane disruption arises from competition for the lipid supply to buds and tubules and an inability to relieve membrane strains. These findings offer a conceptually new model to explain how fatty acid and monoglyceride interactions can trigger phospholipid membrane remodeling events relevant to various biophysical and biological systems.

Abstract A48: Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems

Abstract Background: In cancer research, studying cell motility is fundamental to investigate cancer invasion and drug resistance. In solid tumors constituted by a huge stroma component, such as pancreatic ductal adenocarcinoma (PDAC), the ability to quantify cell movement and interactions is mandatory to better understand the complex crosstalk between cancer and stroma cells. In PDAC, the pancreatic stellate cells (PSCs) are the principal source of dense fibrotic stroma: these closely interact with the PDAC cells to create a facilitating tumor microenvironment that supports local and distant tumor progression through the secretion and/or the response to a number of cytokines that globally increase cancer invasiveness; moreover, the dense tumor microenvironment contributes to resistance to chemotherapy and radiation therapy. Recent studies have established that targeting the stromal compartment in PDAC may lead to promising outcomes. Unfortunately, significant improvements in the overall survival of patients have not been realized in more than four decades, in part because of the lack of relevant preclinical models. Methods: To infer interactions existing between stromal and cancer cells in complex 3D environments, we developed a novel platform that combines time-lapse fluorescence microscopy, automated image detection, and extensive statistical analysis. To better mimic in situ PDAC, we created a hydrogel-based (Matrigel, collagen) model that allows cell tracking in 4 dimensions (x, y, z, t), not possible in 2D, using a mixed population of L3.6PL pancreatic cancer cells and PSCs at a ratio of 25% and 75%, respectively. To precisely identify the two populations and facilitate automatic cell detection, cells were transfected with GFP and mCherry vectors. Once in the 3D conformation, the cocultures were monitored through time-lapse confocal microscopy (CLSM) in controlled conditions, and the dataset containing the temporal evolution of the cells was processed by statistical tools. Results: Key to our approach was the generation of new machine learning-driven automated inferential protocols that resulted in the high-resolution imaging of the strength of the interactions between cells, as well as the potential presence of local chemokine gradients. In our ongoing studies, both commercial PDAC cell lines and pancreatic stellate cells were used to establish the model system. We are currently adopting the model to take full advantage of patient-derived PDAC cells established using the conditionally reprogrammed cells technique, mixed with patient-matched stellate cells. Since cell migration is a hallmark of cancer, we believe that this platform could be used as a reliable and reproducible approach for studying single-cell migration and invasion also in patient-derived models. The research leading to these results received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 759959, ERC-StG “INTERCELLMED”). Citation Format: Marta Maria Cavo, Francesco Alemanno, Donatella Delle Cave, Eliana D'Amone, Adriano Barra, Enza Lonardo, Erika Parasido, Chris Albanese, Loretta Laureana del Mercato. Quantifying stroma-tumor cell interactions in three-dimensional cell culture systems [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A48.

Optimal formation of uniform-phase supported lipid bilayers from phospholipid–monoglyceride bicellar mixtures

Supported lipid bilayers (SLBs) spanning hydrophilic surfaces are industrially attractive biomimetic coatings that mimic critical aspects of lipid membrane interfaces and are increasingly used in applications spanning medicine, biotechnology, and environmental science. The use of adsorbing bicelle lipid nanostructures composed of long- and short-chain phospholipid mixtures is an effective self-assembly driven process for streamlined SLB fabrication. However, existing studies use synthetic short-chain phospholipids as a necessary bicelle component and such materials are not practical for industrial applications. Herein, we investigated optimal conditions to fabricate SLBs from bicelles containing an industrially useful monoglyceride called monocaprin (MC) in place of short-chain phospholipids. The ratio of long-chain phospholipid to MC along with total lipid concentration were systematically tested. Quartz crystal microbalance-dissipation (QCM-D) and time-lapse fluorescence microscopy experiments were performed to characterize bicelle adsorption onto silicon dioxide surfaces, and fluorescence recovery after photobleaching (FRAP) measurements were conducted to evaluate lateral lipid diffusion within the fabricated lipid adlayers. Depending on bicelle parameters, high-quality SLB formation with uniform phase properties was achieved and optimal ranges are described to ensure target performance outcomes without phase separation. Together, our findings demonstrate that MC-containing bicelles are useful tools to form high-quality SLBs suitable for surface coating and biosensing applications.

Supported Lipid Bilayer Formation from Phospholipid-Fatty Acid Bicellar Mixtures.

Supported lipid bilayers (SLBs) are versatile cell membrane-mimicking biointerfaces for various applications such as biosensors and drug delivery systems, and there is broad interest in developing simple, cost-effective methods to achieve SLB fabrication. One promising approach involves the deposition of quasi-two-dimensional bicelle nanostructures that are composed of long-chain phospholipids and either short-chain phospholipids or detergent molecules. While a variety of long-chain phospholipids have been used to prepare bicelles for SLB fabrication applications, only two short-chain phospholipids, 1,2-dihexanoyl-sn-glycero-3-phosphocholine and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (collectively referred to as DHPC), have been investigated. There remains an outstanding need to identify natural alternatives to DHPC, especially ones that are more affordable, to improve fabrication prospects and application opportunities. Herein, we explored the potential to fabricate SLBs from bicellar mixtures composed of long-chain phospholipids and lauric acid (LA), which is a low-cost, naturally abundant fatty acid that is widely used in soapmaking and various industrial applications. Quartz crystal microbalance-dissipation (QCM-D) experiments were conducted to track bicelle adsorption onto silica surfaces as a function of bicelle composition and lipid concentration, along with time-lapse fluorescence microscopy imaging and fluorescence recovery after photobleaching (FRAP) experiments to further characterize lipid adlayer properties. The results identified optimal conditions where it is possible to efficiently form SLBs from LA-containing bicelles at low lipid concentrations while also unraveling mechanistic insights into the bicelle-mediated SLB formation process and verifying that LA-containing bicelles are biocompatible with human cells for surface coating applications.

A novel tracking and analysis system for time-lapse cell imaging of <i>Saccharomyces cerevisiae</i>

Recent studies have revealed that tracking single cells using time-lapse fluorescence microscopy is an optimal tool for spatiotemporal evaluation of proteins of interest. Using this approach with Saccharomyces cerevisiae as a model organism, we previously found that heterochromatin regions involved in epigenetic regulation differ between individual cells. Determining the regularity of this phenomenon requires measurement of spatiotemporal epigenetic-dependent changes in protein levels across more than one generation. In past studies, we conducted these analyses manually to obtain a dendrogram, but this required more than 15 h, even for a single set of microscopic cell images. Thus, in this study, we developed a software-based analysis system to analyze time-lapse cellular images of S. cerevisiae, which allowed automatic generation of a dendrogram from a given set of time-lapse cell images. This approach is divided into two phases: a cell extraction and tracking phase, and an analysis phase. The cell extraction and tracking phase generates a set of necessary information for each cell, such as geometrical properties and the daughter-mother relationships, using image processing-based analysis techniques. Then, based on this information, the analysis phase allows generation of the final dendrogram by analyzing the fluorescent characteristics of each cell. The system is equipped with manual error correction to correct for the inevitable errors that occur in these analyses. The time required to obtain the final dendrograms was drastically reduced from 15 h in manual analysis to 0.8 h using this novel system.

Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae.

All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in the yeast Saccharomyces cerevisiae, which is controlled by the Rho GTPase Cdc42p. The prevailing view of bud emergence does not account for regulation by extrinsic cues. Here, we show that the filamentous growth mitogen activated protein kinase (fMAPK) pathway regulates bud emergence under nutrient-limiting conditions. The fMAPK pathway regulated the expression of polarity targets including the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and active during the period of the cell cycle leading up to bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

Mechanical regulation of photosynthesis in cyanobacteria.

Photosynthetic organisms regulate their responses to many diverse stimuli in an effort to balance light harvesting with utilizable light energy for carbon fixation and growth (source-sink regulation). This balance is critical to prevent the formation of reactive oxygen species that can lead to cell death. However, investigating the molecular mechanisms that underlie the regulation of photosynthesis in cyanobacteria using ensemble-based measurements remains a challenge due to population heterogeneity. Here, to address this problem, we used long-term quantitative time-lapse fluorescence microscopy, transmission electron microscopy, mathematical modelling and genetic manipulation to visualize and analyse the growth and subcellular dynamics of individual wild-type and mutant cyanobacterial cells over multiple generations. We reveal that mechanical confinement of actively growing Synechococcus sp. PCC 7002 cells leads to the physical disassociation of phycobilisomes and energetic decoupling from the photosynthetic reaction centres. We suggest that the mechanical regulation of photosynthesis is a critical failsafe that prevents cell expansion when light and nutrients are plentiful, but when space is limiting. These results imply that cyanobacteria must convert a fraction of the available light energy into mechanical energy to overcome frictional forces in the environment, providing insight into the regulation of photosynthesis and how microorganisms navigate their physical environment.

Ultra-Short Antimicrobial Peptoids Show Propensity for Membrane Activity Against Multi-Drug Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.

Lipid Bicelle Micropatterning Using Chemical Lift-Off Lithography.

Supported lipid membranes are versatile biomimetic coatings for the chemical functionalization of inorganic surfaces. Developing simple and effective fabrication strategies to form supported lipid membranes with micropatterned geometries is a long-standing challenge. Herein, we demonstrate how the combination of chemical lift-off lithography (CLL) and easily prepared lipid bicelle nanostructures can yield micropatterned, supported lipid membranes on gold surfaces with high pattern resolution, conformal character, and biofunctionality. Using CLL, we functionalized gold surfaces with patterned arrays of hydrophilic and hydrophobic self-assembled monolayers (SAMs). Time-lapse fluorescence microscopy imaging revealed that lipid bicelles adsorbed preferentially onto the hydrophilic SAM regions, while there was negligible lipid adsorption onto the hydrophobic SAM regions. Functional receptors could be embedded within the lipid bicelles, which facilitated selective detection of receptor-ligand binding interactions in a model streptavidin-biotin system. Quartz crystal microbalance-dissipation measurements further identified that lipid bicelles adsorb irreversibly and remain intact on top of the hydrophilic SAM regions. Taken together, our findings indicate that lipid bicelles are useful lipid nanostructures for reproducibly assembling micropatterned, supported lipid membranes with precise pattern fidelity.

Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy

We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.

Robotic Cell Printing for Constructing Living Yeast Cell Microarrays in Microfluidic Chips

Living cell microarrays in microfluidic chips allow the non-invasive multiplexed molecular analysis of single cells. Here, we developed a simple and affordable perfusion microfluidic chip containing a living yeast cell array composed of a population of cell variants (green fluorescent protein (GFP)-tagged Saccharomyces cerevisiae clones). We combined mechanical patterning in 102 microwells and robotic piezoelectric cell dispensing in the microwells to construct the cell arrays. Robotic yeast cell dispensing of a yeast collection from a multiwell plate to the microfluidic chip microwells was optimized. The developed microfluidic chip and procedure were validated by observing the growth of GFP-tagged yeast clones that are linked to the cell cycle by time-lapse fluorescence microscopy over a few generations. The developed microfluidic technology has the potential to be easily upscaled to a high-density cell array allowing us to perform dynamic proteomics and localizomics experiments.

Abstract P1-06-05: Image driven biophysical mathematical modeling of multicellular breast tumor spheroids

Abstract Introduction: Conventional 2D monolayer cell culture methods have distinct weaknesses in mimicking in vivo tumors as they ignore major phenotypic physical tumor microenvironment (TME) factors. 3D multicellular tumor spheroid (MTS) systems have been developed to study cancer cell growth in the presence of such TME factors, including cell-cell and cell-extracellular matrix (ECM) mechanical interactions. However, the development of analysis methodologies for these complex culture systems has considerably lagged. We hypothesize that a coupled experimental-computational framework using a microscopy image-driven biophysical mathematical model of MTS systems can accurately estimate phenotypic growth and biophysical TME properties, revealing significantly more mechanistic information than traditional morphometric analysis methods. Materials and Methods: MDA-MB-231 triple-negative breast cancer cells were used to generate spheroids. A single 3D MTS embedded in Collagen I ECM was used as a 3D culture tumor model system as they exhibit cell-cell and cell-ECM interactions that mimic in vivo tumors. Fluorescent fiducial microspheres were placed in the ECM to track displacement induced by the MTS. Time-lapse fluorescence microscopy images were taken of the MTS and microspheres for 72 hours on an automated fluorescent microscope every 12-hours. Images were compiled using a customized fully-automated tiling/stitching/image processing software in MATLAB. Time-lapse images of the MTS and microspheres are used in conjunction with subsequent mathematical modeling analysis for biophysical parameter estimation. We assess MTS using a mathematical model based on two coupled partial differential equations describing growth/motility and mechanical equilibrium in response to cellular-induced forces. Parameters describing cell proliferation rate (k), cell diffusion (D), and cellular mechanical forces (λ) are estimated based on fitting acquired microscopy images to the model via an iterative least-squares parameter estimation. Experiments and model-based analyses were performed in triplicate to define the reproducibility of biophysical parameter estimation. Results and Discussion: In this preliminary study, our modeling framework is able to estimate biophysical parameters of cell diffusion, proliferation rate, and cellular mechanical force in a 3D breast cancer cell culture system of invading MTS embedded in ECM with coefficients of variation across all parameters less than 15% on average. The model predictions accurately represent changes in cellular density and ECM deformation induced by cellular traction forces throughout the observed time series, demonstrating that this model is capable of representing MTS/ECM growth behavior and parameterizing the driving biophysical properties. Conclusions: These results indicate that our imaging-driven mathematical modeling framework for estimating biophysical model parameters has potential to reliably characterize 3D MTS growth and invasion. With reproducibility established, we plan to extend the method to investigate these phenotypic biophysical TME properties in a range of culture and treatment condition interventions. These biophysical properties could be utilized in developing anti-neoplastic drug sensitivity assays to improve cancer therapy by enabling patient-specific estimation/prediction of response to agents and dosing conditions. Acknowledgements: NIH-NCI K25CA204599 and P30CA012197 Citation Format: Haley Brooke Johnson, Emily E. Fannin, Alexandra Thomas, Jared A Weis. Image driven biophysical mathematical modeling of multicellular breast tumor spheroids [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-05.

Single-Cell Analysis on Bacterial Competition between Micro-Colonies

The fate of an isogenic bacterial population is related to single-cell phenotypes. However, the phenotypic heterogeneity of bacteria is complex since it responds to a manifold of biological and environmental factors. It is challenging to study cell-to-cell variations in behavior that are involved in bacterial competition since conventional bulk methods lack sufficient control of the environmental conditions and do not provide single-cell information. Here, microfluidic monolayer chambers (MMC) are designed to couple with time-lapse fluorescence microscopy and are able to provide the necessary spatiotemporal resolution for investigating bacterial competition at a single-cell level. We have been studying the mechanisms that enable one bacterial species to dominate within a small, structured micro-colony, mimicking the onset of infection and/or providing a minimal/toy model of the microbiome. Extended micro-colony cultivation experiments of E.coli strains pxG10sf-GFP and pxG10-mCherry, and co-cultivation experiments of those two strains will be discussed.

Gene expression noise in a complex artificial toxin expression system

Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.

PiQSARS: A pipeline for quantitative and statistical analyses of ratiometric fluorescent biosensors

Genetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline for•Semi-automated treatment of time-lapse fluorescence microscopy images.•Quantification of individual cell signal.•Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.

A Recurrent Neural Network for Particle Tracking in Microscopy Images Using Future Information, Track Hypotheses, and Multiple Detections.

Automatic tracking of particles in time-lapse fluorescence microscopy images is essential for quantifying the dynamic behavior of subcellular structures and virus structures. We introduce a novel particle tracking approach based on a deep recurrent neural network architecture that exploits past and future information in both forward and backward direction. Assignment probabilities are determined jointly across multiple detections, and the probability of missing detections is computed. In addition, existence probabilities are determined by the network to handle track initiation and termination. For correspondence finding, track hypotheses are propagated to future time points so that information at later time points can be used to resolve ambiguities. A handcrafted similarity measure and handcrafted motion features are not necessary. Manually labeled data is not required for network training. We evaluated the performance of our approach using image data of the Particle Tracking Challenge as well as real fluorescence microscopy image sequences of virus structures. It turned out that the proposed approach outperforms previous methods.

C. elegans Runx/CBFβ suppresses POP-1 TCF to convert asymmetric to proliferative division of stem cell-like seam cells.

ABSTRACTA correct balance between proliferative and asymmetric cell divisions underlies normal development, stem cell maintenance and tissue homeostasis. What determines whether cells undergo symmetric or asymmetric cell division is poorly understood. To gain insight into the mechanisms involved, we studied the stem cell-like seam cells in the Caenorhabditis elegans epidermis. Seam cells go through a reproducible pattern of asymmetric divisions, instructed by divergent canonical Wnt/β-catenin signaling, and symmetric divisions that increase the seam cell number. Using time-lapse fluorescence microscopy we observed that symmetric cell divisions maintain asymmetric localization of Wnt/β-catenin pathway components. Our observations, based on lineage-specific knockout and GFP-tagging of endogenous pop-1, support the model that POP-1TCF induces differentiation at a high nuclear level, whereas low nuclear POP-1 promotes seam cell self-renewal. Before symmetric division, the transcriptional regulator RNT-1Runx and cofactor BRO-1CBFβ temporarily bypass Wnt/β-catenin asymmetry by downregulating pop-1 expression. Thereby, RNT-1/BRO-1 appears to render POP-1 below the level required for its repressor function, which converts differentiation into self-renewal. Thus, we found that conserved Runx/CBFβ-type stem cell regulators switch asymmetric to proliferative cell division by opposing TCF-related transcriptional repression.

Inter-Cell Networking Profiling Enables Comprehensive Characterization of Immune-Mediated Activity of Anti-CD38 Therapy through Ex-Vivo Analysis of Multiple Myeloma Patients

Inter-Cell Networking Profiling Enables Comprehensive Characterization of Immune-Mediated Activity of Anti-CD38 Therapy through Ex-Vivo Analysis of Multiple Myeloma Patients

Copper oxide nanoparticles alter cellular morphology via disturbing the actin cytoskeleton dynamics in Arabidopsis roots

Copper oxide nanoparticles (CuO NPs) have severe nano-toxic effects on organisms. Limited data is available on influence of CuO NPs on plant cells. Here, the molecular mechanisms involved in the toxicity of CuO NPs are studied. Exposure to CuO NPs significantly increased copper content in roots (0.062–0.325 mg/g FW), but CuO NPs translocation rates from root to shoot were low (1.1–2.8%). Presented data were significant at p < 0.05 compared to control. CuO NPs inhibited longitudinal growth and promoted transverse growth in root tip cells. However, CuO NPs did not affect the leaf cells, implying that the transfer ability of CuO NPs was weak, and toxicity mainly affected roots. CuO NPs can conjugate with actin protein. The actin cytoskeleton experienced reorganization in the presence of CuO NPs. The longitudinal filamentous actin (F-actin) decreased, and the transverse F-actin increased. CuO NPs inhibited actin polymerization and promoted depolymerization. The behavior of individual F-actin was at steady state with time-lapse under CuO NPs treatment by time-lapse reflection fluorescence (TIRF) microscopy. The growth rate of actin filaments was weakened by CuO NPs. CuO NPs disturbed the subcellular localization of PINs and the gradient of auxin distribution in root tips in an actin-dependent manner. In conclusion, CuO NPs conjugated with actin and disturbed F-actin dynamics, triggering abnormal cell growth in the root tip, and findings provide theoretical basis for further study nano-toxicity in plants.

Effect of Topographical Steps on the Surface Motility of the Bacterium Pseudomonas aeruginosa.

Bacteria traverse surfaces as part of colonizing solids, and it is of interest to hinder this motion to potentially thwart infections in humans. Here, we demonstrate that topographical steps hinder the ability of Pseudomonas aeruginosa PAO1 (P. aeruginosa) to traverse a solid-liquid interface. Using time-lapse fluorescence microscopy and image analysis, we analyzed the motion of P. aeruginosa that were challenged with steps ranging in height from 0.4 to 9.0 μm. Bacterial trajectories are sensitive to the height of the step, the curvature of the step face, and the direction of their motion relative to gravity. When the step height is ≥0.9 μm, which is similar to the cell diameter, there is a reduced probability of the cell crossing the step. For those bacteria that do cross a step, there is a time penalty for crossing steps of height 2-3 μm; this height is similar to the length of the bacterium. For higher steps, the bacteria reorient their cell body while traversing the step riser. Our findings elucidate how topography influences the motion of bacteria and informs strategies for hindering bacterial motion at surfaces.