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

ABSTRACTDengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

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Faculty Opinions recommendation of Asymmetric peptide nanoarray surfaces for studies of single cell polarization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1103555.560550 ◽

2008 ◽

Author(s):

Kevin J Luebke

Keyword(s):

Single Cell ◽

Cell Polarization

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A mathematical model of GTPase pattern formation during single-cell wound repair

Interface Focus ◽

10.1098/rsfs.2016.0032 ◽

2016 ◽

Vol 6 (5) ◽

pp. 20160032 ◽

Cited By ~ 8

Author(s):

William R. Holmes ◽

Adriana E. Golding ◽

William M. Bement ◽

Leah Edelstein-Keshet

Keyword(s):

Single Cell ◽

Wound Repair ◽

Critical Role ◽

Experimental Manipulation ◽

Cell Polarization ◽

Wound Response ◽

Dominant Negative ◽

Nucleotide Exchange ◽

Model Approximation ◽

Positive Feedbacks

Rho GTPases are regulatory proteins whose patterns on the surface of a cell affect cell polarization, cell motility and repair of single-cell wounds. The stereotypical patterns formed by two such proteins, Rho and Cdc42, around laser-injured frog oocytes permit experimental analysis of GTPase activation, inactivation, segregation and crosstalk. Here, we review the development and analysis of a spatial model of GTPase dynamics that describe the formation of concentric zones of Rho and Cdc42 activity around wounds, and describe how this model has provided insights into the roles of the GTPase effector molecules protein kinase C (PKCβ and PKCη) and guanosine nucleotide dissociation inhibitor (GDI) in the wound response. We further demonstrate how the use of a ‘sharp switch’ model approximation in combination with bifurcation analysis can aid mapping the model behaviour in parameter space (approximate results confirmed with numerical simulation methods). Using these methods in combination with experimental manipulation of PKC activity (PKC overexpression (OE) and dominant negative conditions), we have shown that: (i) PKCβ most probably acts by enhancing existing positive feedbacks (from Rho to itself via the guanosine nucleotide exchange factor domain of Abr, and from Cdc42 to itself), (ii) PKCη most probably increases basal rates of inactivation (or possibly decreases basal rates of activation) of Rho and Cdc42, and (iii) the graded distribution of PKCη and its effect on initial Rho activity accounts for inversion of zones in a fraction (20%) of PKCη OE cells. Finally, we speculate that GDIs (which sequester GTPases) may have a critical role in defining the spatial domain, where the wound response may occur. This paper provides a more thorough exposition of the methods of analysis used in the investigation, whereas previous work on this topic was addressed to biologists and abbreviated such discussion.

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Uncovering the Gene Regulatory Networks Underlying Macrophage Polarization Through Comparative Analysis of Bulk and Single-Cell Data

10.1101/2021.01.20.427499 ◽

2021 ◽

Author(s):

Klebea Carvalho ◽

Elisabeth Rebboah ◽

Camden Jansen ◽

Katherine Williams ◽

Andrew Dowey ◽

...

Keyword(s):

Single Cell ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Time Course ◽

Macrophage Polarization ◽

Cell Polarization ◽

Rna Seq ◽

Gene Regulatory ◽

Cell Subpopulations ◽

Cell Data

SummaryGene regulatory networks (GRNs) provide a powerful framework for studying cellular differentiation. However, it is less clear how GRNs encode cellular responses to everyday microenvironmental cues. Macrophages can be polarized and potentially repolarized based on environmental signaling. In order to identify the GRNs that drive macrophage polarization and the heterogeneous single-cell subpopulations that are present in the process, we used a high-resolution time course of bulk and single-cell RNA-seq and ATAC-seq assays of HL-60-derived macrophages polarized towards M1 or M2 over 24 hours. We identified transient M1 and M2 markers, including the main transcription factors that underlie polarization, and subpopulations of naive, transitional, and terminally polarized macrophages. We built bulk and single-cell polarization GRNs to compare the recovered interactions and found that each technology recovered only a subset of known interactions. Our data provide a resource to study the GRN of cellular maturation in response to microenvironmental stimuli in a variety of contexts in homeostasis and disease.

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Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based Microfluidics

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.715408 ◽

2021 ◽

Vol 9 ◽

Author(s):

B. M. Tiemeijer ◽

M. W. D. Sweep ◽

J. J. F. Sleeboom ◽

K. J. Steps ◽

J. F. van Sprang ◽

...

Keyword(s):

Single Cell ◽

High Throughput ◽

Biological Function ◽

Single Cells ◽

Macrophage Polarization ◽

Cell Polarization ◽

Droplet Microfluidics ◽

Cellular Heterogeneity ◽

Adherent Cell ◽

M2 Polarization

Human immune cells intrinsically exist as heterogenous populations. To understand cellular heterogeneity, both cell culture and analysis should be executed with single-cell resolution to eliminate juxtacrine and paracrine interactions, as these can lead to a homogenized cell response, obscuring unique cellular behavior. Droplet microfluidics has emerged as a potent tool to culture and stimulate single cells at high throughput. However, when studying adherent cells at single-cell level, it is imperative to provide a substrate for the cells to adhere to, as suspension culture conditions can negatively affect biological function and behavior. Therefore, we combined a droplet-based microfluidic platform with a thermo-reversible polyisocyanide (PIC) hydrogel, which allowed for robust droplet formation at low temperatures, whilst ensuring catalyzer-free droplet gelation and easy cell recovery after culture for downstream analysis. With this approach, we probed the heterogeneity of highly adherent human macrophages under both pro-inflammatory M1 and anti-inflammatory M2 polarization conditions. We showed that co-encapsulation of multiple cells enhanced cell polarization compared to single cells, indicating that cellular communication is a potent driver of macrophage polarization. Additionally, we highlight that culturing single macrophages in PIC hydrogel droplets displayed higher cell viability and enhanced M2 polarization compared to single macrophages cultured in suspension. Remarkably, combining phenotypical and functional analysis on single cultured macrophages revealed a subset of cells in a persistent M1 state, which were undetectable in conventional bulk cultures. Taken together, combining droplet-based microfluidics with hydrogels is a versatile and powerful tool to study the biological function of adherent cell types at single-cell resolution with high throughput.

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Single-cell transcriptional dynamics of flavivirus infection

eLife ◽

10.7554/elife.32942 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 70

Author(s):

Fabio Zanini ◽

Szu-Yuan Pu ◽

Elena Bekerman ◽

Shirit Einav ◽

Stephen R Quake

Keyword(s):

Single Cell ◽

Membrane Trafficking ◽

Viral Infections ◽

Cultured Cells ◽

Complex Dynamics ◽

Single Cell Level ◽

Cell Level ◽

Transcriptional Dynamics ◽

High Degree ◽

Virus Abundance

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

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Fast single-cell biochemistry: theory, open source microscopy and applications

Methods and Applications in Fluorescence ◽

10.1088/2050-6120/ab3bd2 ◽

2019 ◽

Vol 7 (4) ◽

pp. 044001 ◽

Cited By ~ 5

Author(s):

Andrew L Trinh ◽

Suzan Ber ◽

Annie Howitt ◽

Pablo Oriol Valls ◽

Maximilian W Fries ◽

...

Keyword(s):

Single Cell ◽

Open Source ◽

Cell Biochemistry

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