Single-Cell Analysis in Biotechnology, Systems Biology, and Biocatalysis

Fritzsch, Frederik S. O.; Dusny, Christian; Frick, Oliver; Schmid, Andreas

doi:10.1146/annurev-chembioeng-062011-081056

Cited by 174 publications

(139 citation statements)

References 152 publications

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“…Single cells within a population are, however, likely to differ in their properties. Single-cell analysis so far has largely concentrated on transcripts, genes, secreted molecules, and metabolites (32)(33)(34)(35). Differences between single cells in signaling network properties have been less studied.…”

Section: Discussionmentioning

confidence: 99%

Optical control demonstrates switch-like PIP3 dynamics underlying the initiation of immune cell migration

Karunarathne¹,

Giri²,

Patel

et al. 2013

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

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There is a dearth of approaches to experimentally direct cell migration by continuously varying signal input to a single cell, evoking all possible migratory responses and quantitatively monitoring the cellular and molecular response dynamics. Here we used a visual blue opsin to recruit the endogenous G-protein network that mediates immune cell migration. Specific optical inputs to this optical trigger of signaling helped steer migration in all possible directions with precision. Spectrally selective imaging was used to monitor cellwide phosphatidylinositol (3,4,5)-triphosphate (PIP3), cytoskeletal, and cellular dynamics. A switch-like PIP3 increase at the cell front and a decrease at the back were identified, underlying the decisive migratory response. Migration was initiated at the rapidly increasing switch stage of PIP3 dynamics. This result explains how a migratory cell filters background fluctuations in the intensity of an extracellular signal but responds by initiating directionally sensitive migration to a persistent signal gradient across the cell. A twocompartment computational model incorporating a localized activator that is antagonistic to a diffusible inhibitor was able to simulate the switch-like PIP3 response. It was also able simulate the slow dissipation of PIP3 on signal termination. The ability to independently apply similar signaling inputs to single cells detected two cell populations with distinct thresholds for migration initiation. Overall the optical approach here can be applied to understand G-proteincoupled receptor network control of other cell behaviors.optogenetics | ultrasensitivity A variety of cells sense gradients of chemoattractants and respond by migrating toward increasing concentrations. Migration is central to immune cell function, morphogenesis, cancer cell metastasis, and the life cycle of the social amoeba, Dictyostelium discoideum (1). Cell migration is made up of a characteristic sequence of identifiable cellular events that are governed by G-protein-coupled receptor (GPCR)-driven signaling networks. Although considerable information exists about molecules involved in migration, the challenge is in translating a static map of these molecules into a spatially and temporally dynamic network that orchestrates migratory behavior. Effective methods to probe the basis of network control of migration need to be able to faithfully evoke migratory behavior experimentally and quantitatively monitor response dynamics at the cellular and molecular level. Microfluidic devices and electrical fields have been used to regulate migration and provide insights into the process (2-6). However, there are limitations at present in the ability to direct a series of signaling inputs to a single cell in spatially and temporally complex patterns. Such inputs are essential to continually choreograph the events that constitute the migratory response: initiation, translocation, directional changes, and adaptation. A light-sensitive domain of a plant protein has been inserted into Rac1, a downstrea...

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Section: Discussionmentioning

confidence: 99%

Optical control demonstrates switch-like PIP3 dynamics underlying the initiation of immune cell migration

Karunarathne¹,

Giri²,

Patel

et al. 2013

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

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“…With the emergence of single-cell analysis methods, researchers have gained new insights into the mechanisms and kinetics of single-cell processes in cell biology, systems biology, and clinical biology [5][6][7]. The two most frequently used methods for single-cell analysis are flow cytometry [8] and microscopy.…”

Section: Introductionmentioning

confidence: 99%

Real-time monitoring of immobilized single yeast cells through multifrequency electrical impedance spectroscopy

et al. 2014

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We present a microfluidic device, which enables single cells to be reliably trapped and cultivated while simultaneously being monitored by means of multifrequency electrical impedance spectroscopy (EIS) in the frequency range of 10 kHz-10 MHz. Polystyrene beads were employed to characterize the EIS performance inside the microfluidic device. The results demonstrate that EIS yields a low coefficient of variation in measuring the diameters of captured beads (~0.13 %). Budding yeast, Saccharomyces cerevisiae, was afterwards used as model organism. Single yeast cells were immobilized and measured by means of EIS. The bud growth was monitored through EIS at a temporal resolution of 1 min. The size increment of the bud, which is difficult to determine optically within a short time period, can be clearly detected through EIS signals. The impedance measurements also reflect the changes in position or motion of single yeast cells in the trap. By analyzing the multifrequency EIS data, cell motion could be qualitatively discerned from bud growth. The results demonstrate that single-cell EIS can be used to monitor cell growth, while also detecting potential cell motion in real-time and label-free approach, and that EIS constitutes a sensitive tool for dynamic single-cell analysis.

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“…At this point, we want to emphasize the many comprehensive review articles that discuss the challenges and current approaches of single cell analysis (e.g., Zenobi [5] Fritzsch et al [6], Schmid et al [7], Borland et al [8]). Furthermore, in a recent issue of ABC (406/14) entitled "Multiplex platforms in diagnostics and bioanalytics," the state of the art of those methods is discussed, which are already fit for purpose of measuring at cellular levels.…”

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confidence: 99%