Generating kinetic environments to study dynamic cellular processes in single cells

Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between β-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.

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Activation of Nicotinic Acetylcholine Receptors Augments Calcium Channel-mediated Exocytosis in Rat Pheochromocytoma (PC12) Cells

The Journal of General Physiology ◽

10.1085/jgp.111.2.257 ◽

1998 ◽

Vol 111 (2) ◽

pp. 257-269 ◽

Cited By ~ 16

Author(s):

Amy B. Harkins ◽

Aaron P. Fox

Keyword(s):

Pc12 Cells ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Single Cells ◽

Cellular Functions ◽

Nicotinic Acetylcholine ◽

Cellular Processes ◽

Voltage Dependent ◽

Vesicle Secretion ◽

Ca2 Channels

The functional effect of activating Ca2+-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca2+-permeable nAChRs or with voltage steps to activate voltage-dependent Ca2+ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at −80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd2+ to block voltage-activated Ca2+ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca2+ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca2+ channels alone. Our data suggest that Ca2+ influx via nAChRs affects Ca2+-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca2+ entry pathway underlying Ca2+-dependent cellular processes such as exocytosis.

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Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

10.1101/101477 ◽

2017 ◽

Author(s):

Yodai Takei ◽

Sheel Shah ◽

Sho Harvey ◽

Lei S. Qi ◽

Long Cai

Keyword(s):

Single Cells ◽

Embryonic Stem ◽

Dynamic Imaging ◽

Live Cells ◽

Chromosome Dynamics ◽

Cellular Processes ◽

Sequential Fish ◽

Telomere Dynamics ◽

Subtelomeric Regions

ABSTRACTVisualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call “track first and identify later” for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

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An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

10.1101/2020.09.07.286898 ◽

2020 ◽

Author(s):

Valentin Romanov ◽

Giulia Silvani ◽

Huiyu Zhu ◽

Charles D Cox ◽

Boris Martinac

Keyword(s):

Mechanical Properties ◽

Single Cell ◽

High Throughput ◽

Single Cells ◽

Adherent Cells ◽

Dependent Manner ◽

Cellular Processes ◽

Membrane Cytoskeleton ◽

Change Temperature

ABSTRACTCellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 °C and 37 °C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

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Chemical-Biology-derived in vivo Sensors: Past, Present, and Future

CHIMIA International Journal for Chemistry ◽

10.2533/chimia.2021.1017 ◽

2021 ◽

Vol 75 (12) ◽

pp. 1017-1021

Author(s):

Robbie Loewith ◽

Aurélien Roux ◽

Olivier Pertz

Keyword(s):

Chemical Biology ◽

Single Cells ◽

Molecular Probes ◽

Cell Physiology ◽

Full Potential ◽

Macromolecular Assemblies ◽

Cellular Processes ◽

Spatio Temporal ◽

High Dimensional Datasets

To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology.

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Generalizing RNA velocity to transient cell states through dynamical modeling

10.1101/820936 ◽

2019 ◽

Cited By ~ 58

Author(s):

Volker Bergen ◽

Marius Lange ◽

Stefan Peidli ◽

F. Alexander Wolf ◽

Fabian J. Theis

Keyword(s):

Steady State ◽

Cell Fate ◽

Single Cells ◽

Internal Clock ◽

Mrna Levels ◽

Driver Genes ◽

Cellular Processes ◽

Regulatory Changes ◽

Transcriptional Dynamics ◽

Latent Time

AbstractThe introduction of RNA velocity in single cells has opened up new ways of studying cellular differentiation. The originally proposed framework obtains velocities as the deviation of the observed ratio of spliced and unspliced mRNA from an inferred steady state. Errors in velocity estimates arise if the central assumptions of a common splicing rate and the observation of the full splicing dynamics with steady-state mRNA levels are violated. With scVelo (https://scvelo.org), we address these restrictions by solving the full transcriptional dynamics of splicing kinetics using a likelihood-based dynamical model. This generalizes RNA velocity to a wide variety of systems comprising transient cell states, which are common in development and in response to perturbations. We infer gene-specific rates of transcription, splicing and degradation, and recover the latent time of the underlying cellular processes. This latent time represents the cell’s internal clock and is based only on its transcriptional dynamics. Moreover, scVelo allows us to identify regimes of regulatory changes such as stages of cell fate commitment and, therein, systematically detects putative driver genes. We demonstrate that scVelo enables disentangling heterogeneous subpopulation kinetics with unprecedented resolution in hippocampal dentate gyrus neurogenesis and pancreatic endocrinogenesis. We anticipate that scVelo will greatly facilitate the study of lineage decisions, gene regulation, and pathway activity identification.

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Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy.

The Journal of Cell Biology ◽

10.1083/jcb.76.3.639 ◽

1978 ◽

Vol 76 (3) ◽

pp. 639-651 ◽

Cited By ~ 7

Author(s):

H Gershman ◽

J J Rosen

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Surface Topography ◽

Single Cells ◽

Cell Contact ◽

Intercellular Contact ◽

3T3 Cells ◽

Peripheral Cell ◽

Cellular Processes ◽

Scanning Electron

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.

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Few shot domain adaptation for in situ macromolecule structural classification in cryoelectron tomograms

Bioinformatics ◽

10.1093/bioinformatics/btaa671 ◽

2020 ◽

Author(s):

Liangyong Yu ◽

Ran Li ◽

Xiangrui Zeng ◽

Hongyi Wang ◽

Jie Jin ◽

...

Keyword(s):

Deep Learning ◽

Large Scale ◽

Spatial Organization ◽

Domain Adaptation ◽

Single Cells ◽

Supplementary Information ◽

Target Domain ◽

Source Domain ◽

Cellular Processes ◽

Cross Domain

Abstract Motivation Cryoelectron tomography (cryo-ET) visualizes structure and spatial organization of macromolecules and their interactions with other subcellular components inside single cells in the close-to-native state at submolecular resolution. Such information is critical for the accurate understanding of cellular processes. However, subtomogram classification remains one of the major challenges for the systematic recognition and recovery of the macromolecule structures in cryo-ET because of imaging limits and data quantity. Recently, deep learning has significantly improved the throughput and accuracy of large-scale subtomogram classification. However, often it is difficult to get enough high-quality annotated subtomogram data for supervised training due to the enormous expense of labeling. To tackle this problem, it is beneficial to utilize another already annotated dataset to assist the training process. However, due to the discrepancy of image intensity distribution between source domain and target domain, the model trained on subtomograms in source domain may perform poorly in predicting subtomogram classes in the target domain. Results In this article, we adapt a few shot domain adaptation method for deep learning-based cross-domain subtomogram classification. The essential idea of our method consists of two parts: (i) take full advantage of the distribution of plentiful unlabeled target domain data, and (ii) exploit the correlation between the whole source domain dataset and few labeled target domain data. Experiments conducted on simulated and real datasets show that our method achieves significant improvement on cross domain subtomogram classification compared with baseline methods. Availability and implementation Software is available online https://github.com/xulabs/aitom. Supplementary information Supplementary data are available at Bioinformatics online.

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Stochastic Simulations of Cellular Processes: From Single Cells to Colonies

Computational Systems Biology ◽

10.1016/b978-0-12-405926-9.00013-7 ◽

2014 ◽

pp. 277-293 ◽

Cited By ~ 1

Author(s):

John Cole ◽

Michael J. Hallock ◽

Piyush Labhsetwar ◽

Joseph R. Peterson ◽

John E. Stone ◽

...

Keyword(s):

Single Cells ◽

Stochastic Simulations ◽

Cellular Processes

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Optimum Threshold Minimizes Noise in Timing of Intracellular Events

10.1101/2020.02.14.949891 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sherin Kannoly ◽

Tianhui Gao ◽

Supravat Dey ◽

Ing-Nang Wang ◽

Abhyudai Singh ◽

...

Keyword(s):

Fundamental Problem ◽

Single Cells ◽

Threshold Level ◽

Site Directed Mutagenesis ◽

Critical Threshold ◽

Cellular Processes ◽

Lysis Time ◽

Escherchia Coli ◽

Stochastic Expression ◽

Optimum Threshold

ABSTRACTHow the noisy expression of regulatory proteins affects timing of intracellular events is an intriguing fundamental problem that influences diverse cellular processes. Here we use the bacteriophage λ to study event timing in individual cells where cell lysis is the result of expression and accumulation of a single protein (holin) in the Escherchia coli cell membrane up to a critical threshold level. Site-directed mutagenesis of the holin gene was used to generate phage variants that vary in their timing of lysis from 30 to 190 min. Observation of the lysis times of single cells reveals an intriguing finding – the noise in lysis timing first decreases with increasing lysis time to reach a minimum, and then sharply increases at longer longer lysis times. A mathematical model with stochastic expression of holin together with dilution from cell growth was sufficient to explain the non-monotonic noise profile, and identify holin accumulation thresholds that generate precision in lysis timing.

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