Tunable Single-Cell Extraction for Molecular Analyses

Guillaume‐Gentil, Orane; Grindberg, Rashel V.; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent A.; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A.

doi:10.1016/j.cell.2016.06.025

Cited by 162 publications

(201 citation statements)

References 39 publications

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“…By analyzing single cells, it becomes possible to understand cellular heterogeneity, to make measurements with improved spatial and temporal resolution, and to study processes for which it is impossible or impractical to collect sufficient material for bulk assays. Single-cell sequencing approaches have been widely used (Wang and Navin, 2015), including in C. elegans (Hashimshony et al, 2012; Osborne Nishimura et al, 2015; Tintori et al, 2016); and single-cell enzyme activity measurements have been pioneered (Guillaume-Gentil et al, 2016; Kovarik and Allbritton, 2011). Here, we presented a single-cell approach for measuring protein-protein interactions at the single molecule level in individual, staged C. elegans embryos.…”

Section: Discussionmentioning

confidence: 99%

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization

et al. 2017

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Summary Regulated protein-protein interactions are critical for cell signaling, differentiation and development. To study dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here, we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined timepoints and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during C. elegans zygote polarization, which takes place in less than 20 minutes. PAR complex dynamics are linked to the cell cycle by polo-like kinase 1, and govern movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo.

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Section: Discussionmentioning

confidence: 99%

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization

et al. 2017

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“…Recently, the nano-sampling approach was extended using an atomic force microscopy-based sampling platform with controlled picoliter volume extraction, followed by a single-time point mRNA analysis (27). This significant advance was found to be largely nondestructive, with 86% cell viability, demonstrating that cells may lose a fraction of their volume without apoptosis.…”

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confidence: 99%

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

Cao

Hjort

Chen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

129

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Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nmdiameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.sampling | nanotechnology | molecular biology | cellular biology

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“…This strategy distinguishes the greater decrease in FRET signal for nanoneedles inserted within the cytosol from a smaller decrease associated with nuclear insertion, to provide spatially localized information. The fluidic force microscope (FluidFM) provides a 400nm nanochannel leading to a microfluidic reservoir integrated within a pyramidal AFM tip 80 . The FluidFM can approach and contact individual cells under optical microscopy guidance, and by applying underpressure, it can extract as little as 0.1pl of fluid from a single cell.…”

Section: Sensing Biomoleculesmentioning

confidence: 99%

Nanoneedle-Based Sensing in Biological Systems

Chiappini

2017

ACS Sens.

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Nanoneedles are high aspect ratio nanostructures with a unique biointerface. Thanks to their peculiar yet poorly understood interaction with cells, they very effectively sense intracellular conditions, typically with lower toxicity and perturbation than traditionally available probes. Through long-term, reversible interfacing with cells, nanoneedles can monitor biological functions over the course of several days. Their nanoscale dimension and the assembly into large scale, ordered, dense arrays enable monitoring the functions of large cell populations, to provide functional maps with sub-micron spatial resolution. Intracellularly, they sense electrical activity of complex excitable networks, as well as concentration, function and interaction of biomolecules in situ, while extracellularly they can measure the forces exerted by cells with piconewton detection limits, or efficiently sort rare cells based on their membrane receptors. Nanoneedles can investigate the function of many biological systems, ranging from cells, to biological fluids, to tissues and living organisms. This review examines the devices, strategies and workflows developed to use nanoneedles for sensing in biological systems. KeywordsNanoneedles, nanowires, biointerface, intracellular sensing, review, biomarkers, label-free, minimally invasive.Nanoneedles are rapidly emerging as a tool to interact with the intracellular environment of large number of cells simultaneously, with limited perturbation of their physiological processes. This interaction provides characteristics advantages for minimally invasive cell and molecular biology investigations, as well as to progress biomedical translation of regenerative and precision medicine approaches. A quick string of several successful proofs of principles have established nanoneedles' potential to efficiently deliver impermeant molecules 1,2 and nanoparticles 3 directly to the cell cytosol, and to sense the intracellular milieu 4-6 across biological systems ranging from cells in culture 7 to living organisms 8 . Nanoneedles can be broadly defined as nanomaterials whose high aspect ratio enables their biological interaction; this definition encompasses a broad range of classically defined nanomaterials, which cater for different biointerfacing needs and applications ( Figure 1). Figure 1 Overview of the nanoneedle devices and strategies used for sensing in biological systems.Each nanoneedle is a stacked bar of a bar graph. The legend below each nanoneedle is color-coded and connects to the associated bar. The height of each bar represents the fraction of reviewed papers with that characteristic. The number in each bar is the number of reviewed publications with that characteristic. Numbers for each bar do not sum to the same total as publications can contribute to more than one bar in a given stack. *"Fluid" excludes biological fluids, + "in vivo/ex vivo" includes biological fluids. The overview is compiled from all the publications reviewed herein that include sensing work.Nanoneedles belong t...

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Tunable Single-Cell Extraction for Molecular Analyses

Cited by 162 publications

References 39 publications

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization

A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

Nanoneedle-Based Sensing in Biological Systems

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