Isolating live cells after high-throughput, long-term, time-lapse microscopy

Scott Luro, Laurent Potvin-Trottier, Burak Okumus & Johan Paulsson

Single-cell genetic screens can be incredibly powerful, but current high-throughput platforms do not track dynamic processes, and even for non-dynamic properties they struggle to separate mutants of interest from phenotypic outliers of the wild-type population. Here we introduce SIFT, single-cell isolation following time-lapse imaging, to address these limitations. After imaging and tracking individual bacteria for tens of consecutive generations under tightly controlled growth conditions, cells of interest are isolated and propagated for downstream analysis, free of contamination and without genetic or physiological perturbations. This platform can characterize tens of thousands of cell lineages per day, making it possible to accurately screen complex phenotypes without the need for barcoding or genetic modifications. We applied SIFT to identify a set of ultraprecise synthetic gene oscillators, with circuit variants spanning a 30-fold range of average periods. This revealed novel design principles in synthetic biology and demonstrated the power of SIFT to reliably screen diverse dynamic phenotypes.

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Thermophoretic trap for single amyloid fibril and protein aggregation studies

Martin Fränzl, Tobias Thalheim, Juliane Adler, Daniel Huster, Juliane Posseckardt, Michael Mertig & Frank Cichos

The study of the aggregation of soluble proteins into highly ordered, insoluble amyloid fibrils is fundamental for the understanding of neurodegenerative disorders. Here, we present a method for the observation of single amyloid fibrils that allows the investigation of fibril growth, secondary nucleation or fibril breakup that is typically hidden in the average ensemble. Our approach of thermophoretic trapping and rotational diffusion measurements is demonstrated for single Aβ40, Aβ42 and pyroglutamyl-modified amyloid-β variant (pGlu3-Aβ3–40) amyloid fibrils.

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A comparison of methods to assess cell mechanical properties

Pei-Hsun Wu, Dikla Raz-Ben Aroush, Atef Asnacios, Wei-Chiang Chen, Maxim E. Dokukin, Bryant L. Doss, Pauline Durand-Smet, Andrew Ekpenyong, Jochen Guck, Nataliia V. Guz, Paul A. Janmey, Jerry S. H. Lee, Nicole M. Moore, Albrecht Ott, Yeh-Chuin Poh, Robert Ros, Mathias Sander, Igor Sokolov, Jack R. Staunton, Ning Wang, Graeme Whyte & Denis Wirtz

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

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Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1

Pia Ringer, Andreas Weißl, Anna-Lena Cost, Andrea Freikamp, Benedikt Sabass, Alexander Mehlich, Marc Tramier, Matthias Rief & Carsten Grashoff

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3–5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.

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STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA

Iddo Heller, Gerrit Sitters, Onno D Broekmans, Géraldine Farge, Carolin Menges, Wolfgang Wende, Stefan W Hell, Erwin J G Peterman & Gijs J L Wuite
Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy. Proteins on DNA are visualized at a resolution of 50 nm, a sixfold resolution improvement over that of confocal microscopy. High temporal resolution (<50 ms) is ensured by fast one-dimensional beam scanning. Individual trajectories of proteins translocating on DNA can thus be distinguished and tracked with high precision. We demonstrate our multimodal approach by visualizing the assembly of dense nucleoprotein filaments with unprecedented spatial resolution in real time. Experimental access to the force-dependent kinetics and motility of DNA-associating proteins at biologically relevant protein densities is essential for linking idealized in vitro experiments with the in vivo situation.

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Quantitative optical trapping on single organelles in cell extract

Pradeep Barak, Ashim Rai, Priyanka Rai & Roop Mallik

We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.
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Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

Marco Capitanio, Monica Canepari, Manuela Maffei, Diego Beneventi, Carina Monico, Francesco Vanzi, Roberto Bottinelli & Francesco Saverio Pavone

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ~10 μs, allows detection of interactions as brief as ~100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

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Ultrahigh-resolution optical trap with single-fluorophore sensitivity

Matthew J Comstock, Taekjip Ha & Yann R Chemla

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore–labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).

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High-resolution, long-term characterization of bacterial motility using optical tweezers

Taejin L Min, Patrick J Mears, Lon M Chubiz, Christopher V Rao, Ido Golding & Yann R Chemla

We present a single-cell motility assay, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of 100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we characterized the long-term statistics of the run-tumble time series in individual Escherichia coli cells. We also quantified higher-order features of bacterial swimming, such as changes in velocity and reversals of swimming direction.

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