scholarly journals Chemical operations on a living single cell by open microfluidics for wound repair studies and organelle transport analysis

2019 ◽  
Vol 10 (7) ◽  
pp. 2081-2087 ◽  
Author(s):  
Sifeng Mao ◽  
Qiang Zhang ◽  
Wu Liu ◽  
Qiushi Huang ◽  
Mashooq Khan ◽  
...  
Keyword(s):  
Laminar Flow ◽  
Single Cell ◽  
Wound Repair ◽  
Organelle Transport ◽  
Transport Analysis ◽  
Living Single

We report a laminar flow based approach that is capable of precisely cutting off or treating a portion of a single cell from its remaining portion in its original adherent state.

scholarly journals Microfluidic guillotine for single-cell wound repair studies

2017 ◽  
Vol 114 (28) ◽  
pp. 7283-7288 ◽  
Author(s):  
Lucas R. Blauch ◽  
Ya Gai ◽  
Jian Wei Khor ◽  
Pranidhi Sood ◽  
Wallace F. Marshall ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Wound Repair ◽  
Time Course ◽  
Single Cells ◽  
Repair Process ◽  
Gene Knockdown ◽  
Viscous Stress ◽  
Repair Capacity ◽  
Stentor Coeruleus

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

scholarly journals Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling

2011 ◽  
Vol 193 (3) ◽  
pp. 455-464 ◽  
Author(s):  
Maria Teresa Abreu-Blanco ◽  
Jeffrey M. Verboon ◽  
Susan M. Parkhurst
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Single Cell ◽  
Wound Repair ◽  
Single Cells ◽  
Repair Process ◽  
Wound Edge ◽  
Cytoskeletal Remodeling ◽  
Tissue Integrity ◽  
E Cadherin

When single cells or tissues are injured, the wound must be repaired quickly in order to prevent cell death, loss of tissue integrity, and invasion by microorganisms. We describe Drosophila as a genetically tractable model to dissect the mechanisms of single-cell wound repair. By analyzing the expression and the effects of perturbations of actin, myosin, microtubules, E-cadherin, and the plasma membrane, we define three distinct phases in the repair process—expansion, contraction, and closure—and identify specific components required during each phase. Specifically, plasma membrane mobilization and assembly of a contractile actomyosin ring are required for this process. In addition, E-cadherin accumulates at the wound edge, and wound expansion is excessive in E-cadherin mutants, suggesting a role for E-cadherin in anchoring the actomyosin ring to the plasma membrane. Our results show that single-cell wound repair requires specific spatial and temporal cytoskeleton responses with distinct components and mechanisms required at different stages of the process.

scholarly journals Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming

Cell ◽  
2019 ◽  
Vol 176 (4) ◽  
pp. 928-943.e22 ◽  
Author(s):  
Geoffrey Schiebinger ◽  
Jian Shu ◽  
Marcin Tabaka ◽  
Brian Cleary ◽  
Vidya Subramanian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Optimal Transport ◽  
Developmental Trajectories ◽  
Cell Gene Expression ◽  
Transport Analysis ◽  
Cell Gene

scholarly journals Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity under Homeostatic Laminar Flow

2020 ◽  
Author(s):  
Ziqing Liu ◽  
Dana L Ruter ◽  
Kaitlyn Quigley ◽  
Yuchao Jiang ◽  
Victoria L Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Laminar Flow ◽  
Single Cell ◽  
Rna Sequencing ◽  
Protein Translation ◽  
Flow Conditions ◽  
Expression Levels ◽  
Cell Behaviors ◽  
Single Cell Rna Sequencing

ABSTRACTObjectiveEndothelial cells that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to endothelial cell quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level endothelial cells regulate overall expression heterogeneity during this transition is poorly understood. Here we profiled endothelial cell transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow.Approach and ResultsSingle-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human endothelial cells under homeostatic laminar flow compared to non-flow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous. Analysis of a subset of genes for relative protein expression revealed that most protein profiles showed decreased heterogeneity under flow. In contrast, the magnitude of expression level changes in RNA and protein was coordinated among endothelial cells in flow vs. non-flow conditions.ConclusionsEndothelial cells exposed to homeostatic laminar flow showed increased cohort heterogeneity in RNA expression levels, while cohort expression heterogeneity of selected cognate proteins decreased under laminar flow. These findings suggest that EC homeostasis is imposed at the level of protein translation and/or stability rather than transcriptionally.

scholarly journals Actin Cytoskeletal Dynamics in Single-Cell Wound Repair

2021 ◽  
Vol 22 (19) ◽  
pp. 10886
Author(s):  
Malene Laage Ebstrup ◽  
Catarina Dias ◽  
Anne Sofie Busk Heitmann ◽  
Stine Lauritzen Sønder ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Single Cell ◽  
Wound Repair ◽  
Membrane Integrity ◽  
Limited Attention ◽  
Membrane Repair ◽  
Cortical Actin ◽  
Comprehensive Overview ◽  
Repair Mechanisms

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Micro/extended-nano sampling interface from a living single cell

The Analyst ◽  
2017 ◽  
Vol 142 (10) ◽  
pp. 1689-1696 ◽  
Author(s):  
L. Lin ◽  
K. Mawatari ◽  
K. Morikawa ◽  
Y. Pihosh ◽  
A. Yoshizaki ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Sampling Interface ◽  
Living Single

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells.

scholarly journals Optimal transport analysis reveals trajectories in steady-state systems

2021 ◽  
Vol 17 (12) ◽  
pp. e1009466
Author(s):  
Stephen Zhang ◽  
Anton Afanassiev ◽  
Laura Greenstreet ◽  
Tetsuya Matsumoto ◽  
Geoffrey Schiebinger
Keyword(s):  
Single Cell ◽  
Optimal Transport ◽  
Single Cell Analysis ◽  
Simulated Data ◽  
Unified Approach ◽  
Transport Analysis ◽  
Time Courses ◽  
Cell Trajectories ◽  
Cell Data ◽  
Natural Way

Understanding how cells change their identity and behaviour in living systems is an important question in many fields of biology. The problem of inferring cell trajectories from single-cell measurements has been a major topic in the single-cell analysis community, with different methods developed for equilibrium and non-equilibrium systems (e.g. haematopoeisis vs. embryonic development). We show that optimal transport analysis, a technique originally designed for analysing time-courses, may also be applied to infer cellular trajectories from a single snapshot of a population in equilibrium. Therefore, optimal transport provides a unified approach to inferring trajectories that is applicable to both stationary and non-stationary systems. Our method, StationaryOT, is mathematically motivated in a natural way from the hypothesis of a Waddington’s epigenetic landscape. We implement StationaryOT as a software package and demonstrate its efficacy in applications to simulated data as well as single-cell data from Arabidopsis thaliana root development.

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

2016 ◽  
Vol 6 (5) ◽  
pp. 20160032 ◽  
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.

Electrofluorochromic Imaging Analysis of Glycan Expression on Living Single Cell with Bipolar Electrode Arrays

2021 ◽  
Vol 93 (12) ◽  
pp. 5114-5122
Author(s):  
Zhaoyan Tian ◽  
Yafeng Wu ◽  
Fengying Shao ◽  
Dezhi Tang ◽  
Xiang Qin ◽  
...  
Keyword(s):  
Single Cell ◽  
Bipolar Electrode ◽  
Imaging Analysis ◽  
Electrode Arrays ◽  
Living Single
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