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 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 mDia2 formin selectively interacts with catenins and not E-cadherin to regulate Adherens Junction formation

2019 ◽  
Author(s):  
Yuqi Zhang ◽  
Krista M. Pettee ◽  
Kathryn N. Becker ◽  
Kathryn M. Eisenmann
Keyword(s):  
Single Cell ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Adherens Junctions ◽  
Single Cells ◽  
Critical Role ◽  
Cell Junctions ◽  
Hek 293 ◽  
E Cadherin ◽  
Cell Cell

AbstractBackgroundEpithelial ovarian cancer (EOC) cells disseminate within the peritoneal cavity, in part, via the peritoneal fluid as single cells, clusters, or spheroids. Initial single cell egress from a tumor can involve disruption of cell-cell adhesions as cells are shed from the primary tumor into the peritoneum. In epithelial cells, Adherens Junctions (AJs) are characterized by homotypic linkage of E-cadherins on the plasma membranes of adjacent cells. AJs are anchored to the intracellular actin cytoskeletal network through a complex involving E-cadherin, p120 catenin, β-catenin, and αE-catenin. However, the specific players involved in the interaction between the junctional E-cadherin complex and the underlying F-actin network remains unclear. Recent evidence indicates that mammalian Diaphanous-related (mDia) formins plays a key role in epithelial cell AJ formation and maintenance through generation of linear actin filaments. Binding of αE-catenin to linear F-actin inhibits association of the branched-actin nucleator Arp2/3, while favoring linear F-actin bundling. We previously demonstrated that loss of mDia2 was associated with invasive single cell egress from EOC spheroids through disruption of junctional F-actin.ResultsIn the current study, we now show that mDia2 has a role at adherens junctions (AJs) in EOC OVCA429 cells and human embryonic kidney (HEK) 293 cells through its association with αE-catenin and β-catenin. mDia2 depletion in EOC cells leads to reduction in actin polymerization and disruption of cell-cell junctions with decreased interaction between β-catenin and E-cadherin.ConclusionsOur results support a necessary role for mDia2 in AJ stability in EOC cell monolayers and indicate a critical role for mDia formins in regulating EOC AJs during invasive transitions.

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.

scholarly journals Cell explosions: single cell death triggers an avoidance response in local populations of the ecologically prominent phytoplankton genus Micromonas

2019 ◽  
Author(s):  
Richard Henshaw ◽  
Jonathan Roberts ◽  
Marco Polin
Keyword(s):  
Cell Death ◽  
Avoidance Response ◽  
Single Cell ◽  
Single Cells ◽  
Local Environment ◽  
Warning Signal ◽  
Predator Prey ◽  
A Cell ◽  
Observed Behaviour ◽  
Micromonas Pusilla

The global phytoplankton community, comprised of aquatic photosynthetic organisms, is acknowledged for being responsible for half of the global oxygen production Prominent among these is the pico-eukaryote Micromonas commoda (formally Micromonas pusilla of the genus Micromonas), which can be found in marine and coastal environments across the globe. Cell death of phytoplankton has been identified as contributing to the largest carbon transfers on the planet moving 109 tonnes of carbon in the oceans every day. During a cell death organic matter is released into the local environment which can act as both a food source and a warning signal for nearby organisms. Here we present a novel motility response to single cell death in populations of Micromonas sp., where the death of a single cell releases a chemical patch triggers surrounding cells to escape the immediate affected area. These so-called “burst events” are then modelled and compared with a spherically symmetric diffusing patch which is found to faithfully reproduce the observed behaviour. Finally, laser ablation of single cells reproduces the observed avoidance response, confirming that Micromonas sp. has evolved a specific motility response in order to escape harmful environments for example nearby predator-prey interactions or virus lysis induced cell death.

scholarly journals A practical solution for preserving single cells for RNA sequencing

2017 ◽  
Author(s):  
Moustafa Attar ◽  
Eshita Sharma ◽  
Shuqiang Li ◽  
Claire Bryer ◽  
Laura Cubitt ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Rna Seq ◽  
Rna Integrity ◽  
Tissue Integrity ◽  
Free Amine ◽  
Gene Level ◽  
Single Cell Isolation ◽  
Amine Groups ◽  
Cell Preservation

AbstractThe design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant’s Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3’ bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

scholarly journals Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate

2018 ◽  
Author(s):  
Maximilian W. Fries ◽  
Kalina T. Haas ◽  
Suzan Ber ◽  
John Saganty ◽  
Emma K. Richardson ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Cell ◽  
Cell Fate ◽  
Genetic Heterogeneity ◽  
Cellular Response ◽  
Single Cells ◽  
Caspase Activation ◽  
Cell Fate Decisions ◽  
Network Activation ◽  
Biochemical Imaging

The biochemical activities underlying cell-fate decisions vary profoundly even in genetically identical cells. But such non-genetic heterogeneity remains refractory to current imaging methods, because their capacity to monitor multiple biochemical activities in single living cells over time remains limited1. Here, we deploy a family of newly designed GFP-like sensors (NyxBits) with fast photon-counting electronics and bespoke analytics (NyxSense) in multiplexed biochemical imaging, to define a network determining the fate of single cells exposed to the DNA-damaging drug cisplatin. By simultaneously imaging a tri-nodal network comprising the cell-death proteases Caspase-2, -3 and -92, we reveal unrecognized single-cell heterogeneities in the dynamics and amplitude of caspase activation that signify survival versus cell death via necrosis or apoptosis. Non-genetic heterogeneity in the pattern of caspase activation recapitulates traits of therapy resistance previously ascribed solely to genetic causes3,4. Chemical inhibitors that alter these patterns can modulate in a predictable manner the phenotypic landscape of the cellular response to cisplatin. Thus, multiplexed biochemical imaging reveals cellular populations and biochemical states, invisible to other methods, underlying therapeutic responses to an anticancer drug. Our work develops widely applicable tools to monitor the dynamic activation of biochemical networks at single-cell resolution. It highlights the necessity to resolve patterns of network activation in single cells, rather than the average state of individual nodes, to define, and potentially control, mechanisms underlying cellular decisions in health and disease.

scholarly journals Into the breach: how cells cope with wounds

Open Biology ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 180135 ◽  
Author(s):  
Mitsutoshi Nakamura ◽  
Andrew N. M. Dominguez ◽  
Jacob R. Decker ◽  
Alexander J. Hull ◽  
Jeffrey M. Verboon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Repair Process ◽  
Molecular Techniques ◽  
Membrane Cytoskeleton ◽  
Type Size ◽  
Cortical Cytoskeleton ◽  
Repair Models

Repair of wounds to individual cells is crucial for organisms to survive daily physiological or environmental stresses, as well as pathogen assaults, which disrupt the plasma membrane. Sensing wounds, resealing membranes, closing wounds and remodelling plasma membrane/cortical cytoskeleton are four major steps that are essential to return cells to their pre-wounded states. This process relies on dynamic changes of the membrane/cytoskeleton that are indispensable for carrying out the repairs within tens of minutes. Studies from different cell wound repair models over the last two decades have revealed that the molecular mechanisms of single cell wound repair are very diverse and dependent on wound type, size, and/or species. Interestingly, different repair models have been shown to use similar proteins to achieve the same end result, albeit sometimes by distinctive mechanisms. Recent studies using cutting edge microscopy and molecular techniques are shedding new light on the molecular mechanisms during cellular wound repair. Here, we describe what is currently known about the mechanisms underlying this repair process. In addition, we discuss how the study of cellular wound repair—a powerful and inducible model—can contribute to our understanding of other fundamental biological processes such as cytokinesis, cell migration, cancer metastasis and human diseases.

Abstract 388: High-throughput Single Cell Tracking of Mitochondrial Function in Cardiomyocytes

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Giovanni Fajardo ◽  
Kristi Bezold ◽  
Tobias Meyer ◽  
Daria Mochly-Rosen ◽  
Daniel Bernstein
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Death ◽  
Membrane Potential ◽  
Intracellular Calcium ◽  
Single Cell ◽  
Mitochondrial Function ◽  
Single Cells ◽  
Cell Populations ◽  
Over Time

Mitochondria play a significant role in the regulation of multiple functions in the heart, ranging from metabolism to cell death. Most mitochondrial assays require either isolating the organelle, thus disrupting the intracellular signaling or using intact cells with average measurements for the entire population neglecting the ability to distinguish cell heterogeneity. Here we describe a novel method for tracking single cell mitochondrial function in cell populations over time. Adult mouse myocytes were exposed to the mitochondrial uncoupler FCCP and hydrogen peroxide to induce changes in membrane potential and oxidative stress, respectively. TMRM and mitosox fluorescence were used to quantify mitochondrial membrane potential and ROS production, Fluo-4 fluorescence was used to assess intracellular calcium. Tracking of single cells was performed using Matlab and Imaris software. After FCCP exposure TMRM signal intensity decreased before there was a significant change in myocyte length that led to hypercontracture and cell death within 10 minutes. To better understand the dynamics of mitochondrial function and its relationship with cell death a dose response curve was established for hydrogen peroxide at 100, 500 and 1000 μM. The higher doses of hydrogen peroxide induced hypercontracture faster than lower doses. For further studies 100 μM was used to assess how homogenous the response to hydrogen peroxide was in cell populations. We found 3 distinct populations of cells responding at different times, a population of cells hypercontracted between 7-10 min, another between 10-15 min and a third population only after 15 min. Changes in membrane potential, oxidative stress and intracellular calcium were simultaneously assessed for every single cell during these time points. In addition, given the organized structure of the mitochondria in the myocyte we have adapted our technique to track individual mitochondria to study heterogeneous responses at the single mitochondrion level. This method provides a unique tool to simultaneously assess multiple parameters of mitochondrial function in single cells over time. In doing so, we have unmasked a complex heterogeneity of single cell behavior that is lost in methods than only average cell populations.

scholarly journals Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus

2020 ◽  
Author(s):  
Kevin S. Zhang ◽  
Lucas R. Blauch ◽  
Wesley Huang ◽  
Wallace F. Marshall ◽  
Sindy K. Y. Tang
Keyword(s):  
Wound Healing ◽  
Single Cell ◽  
Wound Repair ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Wound Response ◽  
Synthetic Cell ◽  
Self Repair ◽  
Stentor Coeruleus

AbstractBackgroundWound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus, a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism.ResultsHere we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales and mechanisms of wound repair in Stentor. We found that Stentor requires ∼100 – 1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ∼8 – 80 μm2/s, faster than most other single cells reported in the literature. Further, we observed and characterized three distinct mechanical modes of wound repair in Stentor: contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor, was surprisingly not important for the contraction mode and was of low importance for the others.ConclusionsWhile events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor, and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved.

scholarly journals Prepatterning by RhoGEFs governs Rho GTPase spatiotemporal dynamics during wound repair

2017 ◽  
Vol 216 (12) ◽  
pp. 3959-3969 ◽  
Author(s):  
Mitsutoshi Nakamura ◽  
Jeffrey M. Verboon ◽  
Susan M. Parkhurst
Keyword(s):  
Wound Repair ◽  
Rho Gtpase ◽  
Single Cells ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Spatial And Temporal Patterns ◽  
Cytoskeletal Remodeling ◽  
Guanine Nucleotide Exchange ◽  
Rho Family ◽  
Membrane Resealing

Like tissues, single cells are subjected to continual stresses and damage. As such, cells have a robust wound repair mechanism comprised of dynamic membrane resealing and cortical cytoskeletal remodeling. One group of proteins, the Rho family of small guanosine triphosphatases (GTPases), is critical for this actin and myosin cytoskeletal response in which they form distinct dynamic spatial and temporal patterns/arrays surrounding the wound. A key mechanistic question, then, is how these GTPase arrays are formed. Here, we show that in the Drosophila melanogaster cell wound repair model Rho GTPase arrays form in response to prepatterning by Rho guanine nucleotide exchange factors (RhoGEFs), a family of proteins involved in the activation of small GTPases. Furthermore, we show that Annexin B9, a member of a class of proteins associated with the membrane resealing, is involved in an early, Rho family–independent, actin stabilization that is integral to the formation of one RhoGEF array. Thus, Annexin proteins may link membrane resealing to cytoskeletal remodeling processes in single cell wound repair.

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