Bulk to Individuality: Specifying Plants’ Cellular Functions Through Single-Cell Omics

Real-time single cell analysis is necessary to understand dynamic cellular functions in time and space. Such analyses require the simultaneous measurement of multiple variables in real-time, due to heterogeneity in cellular populations. We report the application of using a micro-environmental chamber on an automatic laser scanning confocal microscope to observe murine macrophage cells in incubation conditions for more than 18 hours. The motorized stage of the microscope was programmed to scan through pre-defined monitoring locations to increase the observation throughput. The acquired images were post-processed to extract the information of each cell. In contrast to current single-cell technologies, such as fluorescence-activated cell sorter (FACS) based systems, the reported architecture records the history of the physiological responses of individual cells.

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Single-Cell Epigenomics Reveals Mechanisms of Cancer Progression

Annual Review of Cancer Biology ◽

10.1146/annurev-cancerbio-070620-094453 ◽

2022 ◽

Vol 6 (1) ◽

Author(s):

Lindsay M. LaFave ◽

Rachel Savage ◽

Jason D. Buenrostro

Keyword(s):

Single Cell ◽

Cancer Progression ◽

Cancer Biology ◽

Annual Review ◽

Publication Date ◽

Cancer Evolution ◽

Regulatory State ◽

Cellular Functions ◽

Cancer Initiation ◽

Gene Regulatory

Cancer initiation is driven by the cooperation between genetic and epigenetic aberrations that disrupt gene regulatory programs critical to maintain specialized cellular functions. After initiation, cells acquire additional genetic and epigenetic alterations influenced by tumor-intrinsic and -extrinsic mechanisms, which increase intratumoral heterogeneity, reshape the cell's underlying gene regulatory network, and promote cancer evolution. Furthermore, environmental or therapeutic insults drive the selection of heterogeneous cell states, with implications for cancer initiation, maintenance, and drug resistance. The advancement of single-cell genomics has begun to uncover the full repertoire of chromatin and gene expression states (cell states) that exist within individual tumors. These single-cell analyses suggest that cells diversify in their regulatory states upon transformation by co-opting damage-induced and nonlineage regulatory programs that can lead to epigenomic plasticity. Here, we review these recent studies related to regulatory state changes in cancer progression and highlight the growing single-cell epigenomics toolkit poised to address unresolved questions in the field. Expected final online publication date for the Annual Review of Cancer Biology, Volume 6 is April 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Multiplexed live-cell profiling with Raman probes

Nature Communications ◽

10.1038/s41467-021-23700-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chen Chen ◽

Zhilun Zhao ◽

Naixin Qian ◽

Shixuan Wei ◽

Fanghao Hu ◽

...

Keyword(s):

Single Cell ◽

Cell Sorting ◽

Cell Biology ◽

Analytical Techniques ◽

Live Cell ◽

Surface Proteins ◽

Cellular Functions ◽

Straightforward Manner ◽

Raman Probe ◽

Single Living

AbstractSingle-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupling it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. This profiling platform is compatible with live-cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.

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Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus

10.1101/2021.12.13.472349 ◽

2021 ◽

Author(s):

Sereina Olivia Sutter ◽

Anouk Lkharrazi ◽

Elisabeth Maria Schraner ◽

Kevin Michaelsen ◽

Anita Felicitas Meier ◽

...

Keyword(s):

Single Cell ◽

Virus Type ◽

Helper Virus ◽

Single Cell Level ◽

Cellular Functions ◽

Cell Level ◽

Structural Reorganization ◽

Adeno Associated Virus ◽

Viral Genomes

Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

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Interfacing Droplet Microfluidics with Antibody Barcodes for Multiplexed Single-Cell Protein Secretion Profiling

Lab on a Chip ◽

10.1039/d1lc00567g ◽

2021 ◽

Author(s):

Tahereh Khajvand ◽

Peifeng Huang ◽

Linmei Li ◽

Mingxia Zhang ◽

Fengjiao Zhu ◽

...

Keyword(s):

Single Cell ◽

Protein Secretion ◽

Single Cells ◽

Single Cell Protein ◽

Droplet Microfluidics ◽

Cell Protein ◽

Cellular Functions ◽

Disease States

Multiplexed protein secretion analysis of single cells is important to understand the heterogeneity of cellular functions and processes in healthy and disease states. However, current single-cell platforms, such as microwell,...

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Single Cell RNA Sequencing in Atherosclerosis Research

Circulation Research ◽

10.1161/circresaha.119.315940 ◽

2020 ◽

Vol 126 (9) ◽

pp. 1112-1126 ◽

Cited By ~ 10

Author(s):

Jesse W. Williams ◽

Holger Winkels ◽

Christopher P. Durant ◽

Konstantin Zaitsev ◽

Yanal Ghosheh ◽

...

Keyword(s):

Single Cell ◽

Biological Diversity ◽

Single Cells ◽

Cell Types ◽

Atherosclerotic Plaques ◽

Molecular Heterogeneity ◽

Cellular Functions ◽

Technological Advances ◽

Technical Guide ◽

Atherosclerosis Research

Technological advances in characterizing molecular heterogeneity at the single cell level have ushered in a deeper understanding of the biological diversity of cells present in tissues including atherosclerotic plaques. New subsets of cells have been discovered among cell types previously considered homogenous. The commercial availability of systems to obtain transcriptomes and matching surface phenotypes from thousands of single cells is rapidly changing our understanding of cell types and lineage identity. Emerging methods to infer cellular functions are beginning to shed new light on the interplay of components involved in multifaceted disease responses, like atherosclerosis. Here, we provide a technical guide for design, implementation, assembly, and interpretations of current single cell transcriptomics approaches from the perspective of employing these tools for advancing cardiovascular disease research.

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Single-Cell Omics: Cellular Functions

Single-Cell Omics ◽

10.1016/b978-0-12-814919-5.00004-x ◽

2019 ◽

pp. 45-59

Author(s):

Vincenzo Sorrenti ◽

Daniela Gabbia ◽

Stefano Fortinguerra ◽

Alessandro Buriani ◽

Maria Carrara

Keyword(s):

Single Cell ◽

Cellular Functions

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Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9

Molecular Biology of the Cell ◽

10.1091/mbc.e19-04-0223 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2838-2855 ◽

Cited By ~ 1

Author(s):

Takahiko Matsuda ◽

Izumi Oinuma

Keyword(s):

Fluorescence Microscopy ◽

Single Cell ◽

Total Internal Reflection ◽

Synaptic Proteins ◽

Cellular Functions ◽

Postsynaptic Protein ◽

Spatiotemporal Regulation ◽

Dendritic Process ◽

Endogenous Loci ◽

Presynaptic Protein

Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

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Modeling metabolic variation with single-cell expression data

10.1101/2020.01.28.923680 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yuanchao Zhang ◽

Man S. Kim ◽

Elizabeth Nguyen ◽

Deanne M. Taylor

Keyword(s):

Single Cell ◽

Cellular Metabolism ◽

Expression Data ◽

Rna Seq ◽

Cellular Functions ◽

Significant Linear Correlation ◽

Mouse Tissues ◽

Metabolic Variation ◽

Context Specific ◽

Cell Expression

AbstractCellular metabolism encompasses the biochemical reactions and transportation of various metabolites in cells and their surroundings, which are integrated at all levels of cellular functions. We developed a method to systematically simulate cellular metabolism using single-cell RNA-seq (scRNA-seq) data through constraint-based context specific metabolic modeling. We simulated the NAD+ biosynthesis activity in 7 different mouse tissues, and the simulated NAD+ biosynthesis flux levels showed significant linear correlation with experimental measurements in previous research. We also show that the simulated NAD+ biosynthesis fluxes are reproducible using two additional scRNA-seq datasets.

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Multiplexed Live-Cell Profiling with Raman probes

10.21203/rs.3.pex-1507/v1 ◽

2021 ◽

Author(s):

Chen Chen ◽

Zhilun Zhao ◽

Naixin Qian ◽

Shixuan Wei ◽

Fanghao hu ◽

...

Keyword(s):

Single Cell ◽

Cell Biology ◽

Analytical Techniques ◽

Live Cell ◽

Surface Proteins ◽

Cellular Functions ◽

Biological Targets ◽

Systematic Understanding ◽

Raman Probe ◽

Single Living

Abstract Single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understanding of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a super-multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupled it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. Being the highest Raman-based multiplexing technology of biological targets so far, this profiling platform is compatible with live cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.

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