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

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.

Download Full-text

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.

Download Full-text

An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics

10.1101/2021.03.09.434608 ◽

2021 ◽

Author(s):

Caitlin E. T. Donahue ◽

Michael D. Siroky ◽

Katharine A. White

Keyword(s):

Single Cell ◽

Intracellular Ph ◽

Single Cells ◽

Cell Polarization ◽

Membrane Dynamics ◽

Cell Physiology ◽

Single Cell Level ◽

Cell Level ◽

Cell Behaviors ◽

Membrane Ruffling

AbstractIntracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating the experiments required to determine if increased pHi is a driver of these cell behaviors at the single-cell level.

Download Full-text

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.

Download Full-text

SMRT analysis of MTOC and nuclear positioning reveals the role of EB1 and LIC1 in single-cell polarization

Journal of Cell Science ◽

10.1242/jcs.091231 ◽

2011 ◽

Vol 124 (24) ◽

pp. 4267-4285 ◽

Cited By ~ 30

Author(s):

Christopher M. Hale ◽

Wei-Chiang Chen ◽

Shyam B. Khatau ◽

Brian R. Daniels ◽

Jerry S. H. Lee ◽

...

Keyword(s):

Single Cell ◽

Cell Polarization ◽

Nuclear Positioning

Download Full-text

Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo

eLife ◽

10.7554/elife.05279 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 28

Author(s):

Dana Meyen ◽

Katsiaryna Tarbashevich ◽

Torsten U Banisch ◽

Carolina Wittwer ◽

Michal Reichman-Fried ◽

...

Keyword(s):

Cell Migration ◽

Single Cell ◽

Primordial Germ Cells ◽

Cell Polarization ◽

In Vivo Model ◽

Guidance Cue ◽

Rac1 Activity ◽

Cell Front ◽

First Time

Cell migration and polarization is controlled by signals in the environment. Migrating cells typically form filopodia that extend from the cell surface, but the precise function of these structures in cell polarization and guided migration is poorly understood. Using the in vivo model of zebrafish primordial germ cells for studying chemokine-directed single cell migration, we show that filopodia distribution and their dynamics are dictated by the gradient of the chemokine Cxcl12a. By specifically interfering with filopodia formation, we demonstrate for the first time that these protrusions play an important role in cell polarization by Cxcl12a, as manifested by elevation of intracellular pH and Rac1 activity at the cell front. The establishment of this polarity is at the basis of effective cell migration towards the target. Together, we show that filopodia allow the interpretation of the chemotactic gradient in vivo by directing single-cell polarization in response to the guidance cue.

Download Full-text

Dental Pulp Stem Cell Polarization: Effects of Biophysical Factors

Journal of Dental Research ◽

10.1177/00220345211028850 ◽

2021 ◽

pp. 002203452110288

Author(s):

B. Chang ◽

C. Ma ◽

J. Feng ◽

K.K.H. Svoboda ◽

X. Liu

Keyword(s):

Single Cell ◽

Dental Pulp ◽

Underlying Mechanism ◽

Cell Polarization ◽

Limited Effect ◽

3 Dimensional ◽

Biophysical Factors ◽

Dental Pulp Stem Cell ◽

Auxiliary Factor ◽

Dentin Regeneration

Dental pulp stem cells (DPSCs) have the potential to polarize, differentiate, and form tubular dentin under certain conditions. However, the factors that initiate and regulate DPSC polarization and its underlying mechanism remain unclear. Identification of the factors that control DPSC polarization is a prerequisite for tubular dentin regeneration. We recently developed a unique bioinspired 3-dimensional platform that is capable of deciphering the factors that initiate and modulate cell polarization. The bioinspired platform has a simple background and confines a single cell on each microisland of the platform; therefore, it is an effective tool to study DPSC polarization at the single-cell level. In this work, we explored the effects of biophysical factors (surface topography, microisland area, geometry, tubular size, and gravity) on single DPSC polarization. Our results demonstrated that nanofibrous architecture, microisland area, tubular size, and gravity participated in regulating DPSC polarization by influencing the formation of the DPSC process and relocation of the Golgi apparatus. Among these factors, nanofibrous architecture, tubular size, and appropriate microisland area were indispensable for initiating DPSC polarization, whereas gravity served as an auxiliary factor to the process of DPSC polarization. Meanwhile, microisland geometry had a limited effect on DPSC polarization. Collectively, this work provides information on DPSC polarization and paves the way for the development of new biomaterials for tubular dentin regeneration.

Download Full-text