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.

scholarly journals Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5

2008 ◽  
Vol 19 (7) ◽  
pp. 2718-2728 ◽  
Author(s):  
Irfan J. Lodhi ◽  
Dave Bridges ◽  
Shian-Huey Chiang ◽  
Yanling Zhang ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Critical Role ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Glut4 Translocation ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain–containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.

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 Effects of cell tension on the small GTPase Rac

2002 ◽  
Vol 158 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Akira Katsumi ◽  
Julie Milanini ◽  
William B. Kiosses ◽  
Miguel A. del Pozo ◽  
Roland Kaunas ◽  
...  
Keyword(s):  
Critical Role ◽  
Cell Movement ◽  
Cell Polarization ◽  
Mechanical Stresses ◽  
Small Gtpase ◽  
The Body ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Biochemical Signals ◽  
Constitutively Active

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.

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

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
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

Abstract Background Wound 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. Results Here we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response 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 characterized three distinct mechanical modes of wound response 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. Conclusions While 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.

TESTING GOAL-DRIVEN CAPTURE BY THREAT

2019 ◽  
Author(s):  
Chris Robert Harrison Brown
Keyword(s):  
Experimental Evidence ◽  
Attentional Capture ◽  
Critical Role ◽  
Experimental Manipulation ◽  
Contingent Capture ◽  
Top Down ◽  
Affective Stimuli ◽  
Clinical Disorders ◽  
Positive Stimuli

Attention has long been characterised within prominent models as reflecting a competition between goal-driven and stimulus-driven processes. It remains unclear, however, how involuntary attentional capture by affective stimuli, such as threat-laden content, fits into such models. While such effects were traditionally held to reflect stimulus-driven processes, recent research has increasingly implicated a critical role of goal-driven processes. Here we test an alternative goal-driven account of involuntary attentional capture by threat, using an experimental manipulation of goal-driven attention. To this end we combined the classic ‘contingent capture’ and ‘emotion-induced blink’ (EIB) paradigms in an RSVP task with both positive or threatening target search goals. Across six experiments, positive and threat distractors were presented in peripheral, parafoveal, and central locations. Across all distractor locations, we found that involuntary attentional capture by irrelevant threatening distractors could be induced via the adoption of a search goal for a threatening category; adopting a goal for a positive category conversely led to capture only by positive stimuli. Our findings provide direct experimental evidence for a causal role of voluntary goals in involuntary capture by irrelevant threat stimuli, and hence demonstrate the plausibility of a top-down account of this phenomenon. We discuss the implications of these findings in relation to current cognitive models of attention and clinical disorders.

scholarly journals DDRE-06. CELLULAR STRESS RESPONSE IN DIPG THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii62-ii62
Author(s):  
Sreepradha Sridharan ◽  
Arif Harmanci ◽  
Robert Siddaway ◽  
Tara Dobson ◽  
Jyothishmathi Swaminathan ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Synthetic Lethality ◽  
Clonal Evolution ◽  
Single Agent ◽  
Objective Response ◽  
Hdac Inhibitors ◽  
Pediatric Brain Tumor ◽  
Dominant Negative ◽  
Cellular Stress Response

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor of the pons and brainstem. Therefore, there is a desperate need for new therapeutics. Genomic profiling of tumors identified a highly prevalent dominant negative somatic mutation at lysine (K)-27 in histone genes HIST1H3B and H3F3A. Clonal evolution modeling suggests these mutations are truncal, and studies have demonstrated their contribution to tumorigenesis. ONC201, a first-in-class DRD2 antagonist and ClpP agonist is an anticancer drug developed by Oncoceutics, which targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling and is actively being investigated in patients with recurrent H3 K27M-mutant gliomas. In adults with recurrent glioma, single agent studies showed benign-safety, no dose-limiting toxicities and a durable objective response when administered orally. In addition, intra-tumoral drug levels exceeded therapeutic thresholds, and induced tumor cell apoptosis. Based on this and response seen in a pediatric patient with DIPG for whom compassionate use of ONC201 was approved, a multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) is actively accruing patients. However, the strength of UPR and ISR in DIPGs and their effect on DIPG response to ONC201 is not known. Our group employed bulk/single cell transcriptomic and single cell proteomic approaches to demonstrate substantial heterogeneity in UPR and ISR signaling in human DIPG samples. Consistent with this, DIPG cell lines exhibited considerable variability in sensitivity to ONC201. Single cell profiling identified tumor sub-populations with significant proliferative capacity even after ONC201 exposure. Incomplete response promotes recurrence. To target these cells, we performed a synthetic lethality screen with a library of 360 FDA-approved CNS penetrant compounds, which identified HDAC inhibitors and DNA damage-inducing chemotherapy as having synergy with ONC201. Thus, we suggest that tumor heterogeneity impacts sensitivity to ONC201 and that this can be reduced by combination treatments.

scholarly journals Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2089 ◽  
Author(s):  
Iker Lamas ◽  
Nathalie Weber ◽  
Sophie G. Martin
Keyword(s):  
Fission Yeast ◽  
Positive Feedback ◽  
Antagonistic Activity ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Cell Architecture

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

scholarly journals Rapid Development of Th2 Activity During T Cell Priming

2003 ◽  
Vol 10 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Adam F. Cunningham ◽  
Kai-Michael Toellner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rapid Development ◽  
Critical Role ◽  
Cell Polarization ◽  
Th2 Cells ◽  
Th1 Cells ◽  
T Helper ◽  
Th 1 ◽  
Th1 And Th2

The paradigm of T helper-1 (Th-1) and Th-2 cells developing from non-committed naïve precursors is firmly established. Th1 cells are characterized by IFN production and, in mice, the selective switching to IgG2a. Conversely IL-4 production and selective switching to IgG1 and IgE characterize Th2 cells. Analysis of Th2 inductionin vitroindicates that this polarization develops gradually in T cells activated by anti-CD3 in the presence of IL-4; conversely anti-CD3 and IFN induce Th1 cells. In this report, we explore evidence that indicates that the T helper cell polarizationin vivocannot solely be explained by the cytokine environment. This is provided by studying the early acquisition of Th1 and Th2 activities during responses to a mixture of Th1 and Th2-inducing antigens. It is shown that these divergent forms of T cell help can rapidly develop in cells within a single lymph node. It is argued that early polarization to show Th-1 or Th-2 behavior can be induced by signals delivered during cognate interaction between virgin T cells and dendritic cells, in the absence of type 1 or type 2 cytokines. This contrasts with the critical role of the cytokines in reinforcing the Th-phenotype and selectively expanding T helper clones.

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

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