A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death

Murschhauser, Alexandra; Röttgermann, Peter J. F.; Woschée, Daniel; Ober, Martina F.; Yan, Yan; Dawson, Kenneth A.; Rädler, Joachim O.

doi:10.1038/s42003-019-0282-0

Cited by 20 publications

(16 citation statements)

References 45 publications

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“…We believe that fluorescence of the Cy5 labeled mRNAs is unquenched during the time of uptake due to unpacking of the lipid-based carriers. Other studies also used multiple fluorescent signals in single cell time lapse measurement to exploit signal correlations (36,37). Our findings are in agreement with a study by Yasar et al showing that the kinetics of mRNA nanocarrier binding on the cell surface is the same for transfected and nontransfected cells without giving further insights on transfection efficiency (26).…”

Section: Discussionsupporting

confidence: 91%

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Reiser

Woschée

Mehrotra

et al. 2019

Preprint

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Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomalrelease bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticles (LNPs) mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery. author/funder. All rights reserved. No reuse allowed without permission.

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Section: Discussionsupporting

confidence: 91%

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Reiser

Woschée

Mehrotra

et al. 2019

Preprint

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“…Bulk-based studies fail to address the functional heterogeneity underlying a given cell population by masking the phenotype, gene expression, and the mechanism of cellular communication in between individual immune cells 11 , 12 . To overcome the limitations of bulk methodologies, the study of cytotoxicity at a single-cell level can be performed using flow cytometry-based assays and single cell microscopy assays 13 , 14 . However, flow cytometric assays are snapshot-based and therefore not suited to monitor temporal dynamics of cellular interactions leading to cytotoxicity 10 .…”

Section: Introductionmentioning

confidence: 99%

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip

Subedi

Eyndhoven

Hokke

et al. 2021

Sci Rep

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Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.

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“…Additional studies suggested that cytosolic K + depletion by nigericin alone is a minimal common cellular event that is sufficient to activate the NLRP3 inflammasome [ 14 , 17 ]. Other studies have proposed a role for particle-induced lysosomal membrane permeability (LMP) in inflammasome activation and cell death [ 11 , 18 , 19 ]. However, the exact underlying mechanisms leading to LMP have not been described and determination of whether there is any relationship between LMP and changes in cytosolic [K + ] remain to be clarified.…”

Section: Introductionmentioning

confidence: 99%

Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization

Ziglari

Wang

Holian

2021

IJMS

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Lysosomal membrane permeabilization (LMP) has been proposed to precede nanoparticle-induced macrophage injury and NLRP3 inflammasome activation; however, the underlying mechanism(s) of LMP is unknown. We propose that nanoparticle-induced lysosomal hyperpolarization triggers LMP. In this study, a rapid non-invasive method was used to measure changes in lysosomal membrane potential of murine alveolar macrophages (AM) in response to a series of nanoparticles (ZnO, TiO2, and CeO2). Crystalline SiO2 (micron-sized) was used as a positive control. Changes in cytosolic potassium were measured using Asante potassium green 2. The results demonstrated that ZnO or SiO2 hyperpolarized the lysosomal membrane and decreased cytosolic potassium, suggesting increased lysosome permeability to potassium. Time-course experiments revealed that lysosomal hyperpolarization was an early event leading to LMP, NLRP3 activation, and cell death. In contrast, TiO2- or valinomycin-treated AM did not cause LMP unless high doses led to lysosomal hyperpolarization. Neither lysosomal hyperpolarization nor LMP was observed in CeO2-treated AM. These results suggested that a threshold of lysosomal membrane potential must be exceeded to cause LMP. Furthermore, inhibition of lysosomal hyperpolarization with Bafilomycin A1 blocked LMP and NLRP3 activation, suggesting a causal relation between lysosomal hyperpolarization and LMP.

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A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death

Cited by 20 publications

References 45 publications

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

Correlation of mRNA delivery timing and protein expression in lipid-based transfection

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip

Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization

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