Eph-mediated tyrosine phosphorylation of citron kinase controls abscission

Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis.

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Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.191.3.541 ◽

2000 ◽

Vol 191 (3) ◽

pp. 541-550 ◽

Cited By ~ 144

Author(s):

Zhengbin Lu ◽

Lingxian Yuan ◽

Xianzheng Zhou ◽

Eduardo Sotomayor ◽

Hyam I. Levitsky ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cytotoxic T Lymphocyte ◽

Cell Communication ◽

Cd8 T Cell ◽

Cd4 Help ◽

T Cell Help

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.

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SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

Infection and Immunity ◽

10.1128/iai.00064-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 2890-2901 ◽

Cited By ~ 11

Author(s):

Marilena Gallotta ◽

Giovanni Gancitano ◽

Giampiero Pietrocola ◽

Marirosa Mora ◽

Alfredo Pezzicoli ◽

...

Keyword(s):

Cell Division ◽

Mammalian Cells ◽

Group A Streptococcus ◽

Host Cells ◽

Knockout Mutant ◽

Content Type ◽

Moonlighting Protein ◽

Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

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Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2359-2366.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2359-2366

Author(s):

D K Morrison ◽

D R Kaplan ◽

S G Rhee ◽

L T Williams

Keyword(s):

Growth Factor ◽

Tyrosine Phosphorylation ◽

Serine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Pdgf Receptor ◽

Wild Type ◽

Receptor Proteins ◽

Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

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Abstract 12231: PECAM1 is Essential for Flow-mediated Gab1 Tyrosine Phosphorylation and Signaling in vitro and in vivo

Circulation ◽

10.1161/circ.132.suppl_3.12231 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Suowen Xu ◽

Marina Koroleva ◽

Keigi Fujiwara ◽

Zheng Gen Jin

Keyword(s):

Tyrosine Phosphorylation ◽

Wheel Running ◽

Tyrosine Phosphatase ◽

Common Mechanism ◽

Membrane Localization ◽

No Production ◽

Voluntary Wheel Running ◽

Voluntary Wheel

Introduction: Impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued NO production is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Specific signaling cascades, generated by vascular endothelial cells (ECs) in response to laminar flow, modulate EC structure and functions, NO production in particular. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation. However, the upstream mechanism that regulates Gab1 tyrosine phosphorylation remains unclear. Hypothesis: We hypothesized that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Methods: Western blot, en face staining and voluntary wheel running. Results: Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in a flow signaling pathway as well as HGF-induced signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K by LY294002 decreased flow, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. Conclusions: These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs

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Abstract 17071: Cell Communication Inference in Macrophages

Circulation ◽

10.1161/circ.142.suppl_3.17071 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Nika Taghdiri ◽

Kevin R King ◽

David Calcagno ◽

Zhenxing Fu ◽

Kenneth Huang ◽

...

Keyword(s):

Image Analysis ◽

Analytical Methods ◽

Cell Injury ◽

Diffusion Mechanism ◽

Cell Communication ◽

In Vivo Studies ◽

Cell Dynamics ◽

Calcium Indicator

Introduction: Tissue macrophages play diverse roles in the cardiovascular system during health and disease. They have diverse functions within tissues, but our understanding of their dynamics is limited because most macrophage characterization assays are destructive and have low temporal resolution. We asked whether these cells are dynamic and interconnected. Methods: Here, we describe experimental and analytical methods for measuring cell dynamics and inferring communication between cells in vitro and in vivo. We created a mouse (Csf1r-Cre x GCaMP5) expressing the Cre-inducible genetically encoded calcium indicator GCaMP5 under the regulation of the innate immune promoter, Csf1r, to non-destructively quantify high-frequency cell dynamics and differentiated them in culture using m-CSF. We developed custom image analysis routines and parameterization strategies for classifying calcium responses. Results: Our studies revealed that calcium reporter BMDMs display minimal fluctuations at baseline but exhibit a dynamic response to immunogenic DNA sensing. DNA-induced isolated cell injury and death, which precipitated cell communication that spread with a velocity of [9μm/s], consistent with an extracellular diffusion mechanism. We developed quantitative image analysis methods that corrected for random calcium fluctuations and identified statistically significant areas of correlated calcium changes suggestive of communication. An analytical pipeline enabled quantification of calcium spike dynamics and correlations of dynamic calcium profiles of single cell sharing a local microenvironment. This resulted in an “improbable synchrony” metric that allowed localization of communication in time and space. We adapted the pipeline for in vivo studies and tested them in a dorsal window chamber model using intravital microscopy. At 2Hz sampling frequency, we identified 27 potential communication events as they responded to complex microenvironmental cues in vivo. Conclusion: The experimental and analytical methods for inferring cell communication provide a new quantitative toolkit for investigating known as-yet undiscovered cell communication pathways.

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Synthesis of Novel Pyran Fragments to Incorporate into Peloruside Analogues

10.26686/wgtn.17136047.v1 ◽

2021 ◽

Author(s):

◽

Oliver Bayley

Keyword(s):

Cell Division ◽

Scale Up ◽

Great Promise ◽

Peloruside A ◽

Bioactive Secondary Metabolite ◽

Mycale Hentscheli ◽

Significant Attention ◽

Simple Carbohydrate

<p>Cancer is currently the second largest cause of death globally, leading to a high demand for new and effective chemotherapeutics. For years, natural products have been used as a source of new bioactive compounds; of particular interest in this context, as a source of new chemotherapeutics. One chemotherapeutic candidate which has attracted significant attention in synthetic and medicinal chemistry communities, is peloruside A. Peloruside A is a bioactive secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. Since its discovery, peloruside A has shown great promise in cancer studies both in vivo and in vitro with effects observed even at nanomolar concentrations. These chemotherapeutic effects have been shown to occur by halting cell division at the G2/M checkpoint via microtubule stabilisation. Of particular interest is that this stabilisation occurs in a manner distinct from that of the already established taxane class of microtubule stabilising drugs. This means that peloruside A is able to offer both inhibition of cell division in Taxol® resistant cells and synergistic inhibition alongside the current taxane drugs. Since peloruside A is not abundantly available from its natural source, there is a strong incentive for the development of new synthetic strategies for peloruside A production. Unfortunately attempts at aquaculture and attempts at developing an industrial scale synthesis have both proven unsuccessful thus far. In an attempt to overcome some of the difficulties with the scale up of peloruside, analogues have been developed that are intended to have similar bioactivity to peloruside A but simpler, more concise, synthetic routes. These analogues will also enable further elucidation of the binding properties of peloruside A. This project focuses on the generation of a functionalised pyran fragment, starting from a simple carbohydrate, that may be incorporated into the proposed analogues.</p>

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Microfluidic and Lab-on-a-Chip Systems for Cutaneous Wound Healing Studies

10.20944/preprints202104.0703.v1 ◽

2021 ◽

Author(s):

Ghazal Shabestani Monfared ◽

Peter Ertl ◽

Mario Rothbauer

Keyword(s):

Wound Healing ◽

Molecular Mechanisms ◽

Chronic Wounds ◽

Cell Communication ◽

Lab On A Chip ◽

Tissue Level ◽

Cutaneous Wound Healing ◽

Cutaneous Wound

Cutaneous wound healing is a complex multi-stage process involving direct and indirect cell communication events with the aim of efficiently restoring the barrier function of the skin. One key aspect in cutaneous wound healing is associated with cell movement and migration into the physically, chemically and biologically injured area resulting in wound closure. Understanding the conditions under which cell migration is impaired and elucidating the cellular and molecular mechanisms that improve healing dynamics is therefore crucial in devising novel therapeutic strategies to elevate patient suffering, reduce scaring and eliminate chronic wounds. Following the global trend towards automation, miniaturization and integration of cell-based assays into microphysiological systems, conventional wound healing assays such as the scratch assay or cell exclusion assay have recently been translated and improved using microfluidics and lab-on-a-chip technologies. These miniaturized cell analysis systems allow precise spatial and temporal control over a range of dynamic microenvironmental factors including shear stress, biochemical and oxygen gradients to create more reliable in vitro models that resemble the in vivo microenvironment of a wound more closely on a molecular, cellular, and tissue level. The current review provides (a) an overview on the main molecular and cellular processes that take place during wound healing, (b) a brief introduction into conventional in vitro wound healing assays, and (c) a perspective on future cutaneous and vascular wound healing research using microfluidic technology.

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TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

10.1101/2021.02.22.431443 ◽

2021 ◽

Author(s):

Justin V. Joseph ◽

Capucine R. Magaut ◽

Simon Storevik ◽

Luiz H. Geraldo ◽

Thomas Mathivet ◽

...

Keyword(s):

Signaling Pathways ◽

Network Formation ◽

Cell Communication ◽

Treatment Resistance ◽

Communication Structures ◽

Oligodendroglial Tumors ◽

Thrombospondin 1 ◽

Stem Cell Lines

AbstractMicrotubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. To identify potential signaling pathways involved in MT formation we performed a bioinformatics analysis of TCGA data showing that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. In particular we observed that signaling pathways involved in extracellular matrix organization are differentially expressed between these tumor entities. Using patient-derived GBM stem cell lines, we demonstrated that TGF-β1 stimulation promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. In conclusion, TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.

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Abstract 20531: C-Abl Mediated Tyrosine Phosphorylation of Paxillin is Essential for LPS-Induced Endothelial Barrier Dysfunction and Acute Lung Injury

Circulation ◽

10.1161/circ.130.suppl_2.20531 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Panfeng Fu ◽

Anne E Cress ◽

Ting Wang ◽

Joe G Garcia ◽

Viswanathan Natarajan

Keyword(s):

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Human Lung ◽

Adherens Junctions ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Lps Challenge

Paxillin, a multi-domain scaffold-adapter focal adhesion (FA) protein, plays an important role in facilitating protein networking and efficient signaling transduction. Paxillin is phosphorylated at multiple serine/threonine and tyrosine residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and the acute respiratory distress syndrome (ARDS) remains unclear. In this study, we used paxillin-specific siRNA and site-specific non-phosphorylatable mutants of paxillin to abrogate the function of paxillin, both in vitro and in vivo, to determine its role in the regulation of lung endothelial permeability and ARDS. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (ECs) resulted in paxillin accumulation at focal adhesions, enhanced tyrosine phosphorylation of paxillin at Y31 and Y118, and significant endothelial barrier dysfunction. However no significant changes in Y181 phosphorylation by LPS challenge was observed. Paxillin silencing (siRNA) attenuated LPS-induced endothelial barrier dysfunction and dissociation of VE-cadherin from adherens junctions. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase and not by Src or focal adhesion kinase (FAK) in human lung microvascular ECs. Furthermore, down-regulation of c-Abl (siRNA) significantly reduced LPS-mediated endothelial barrier dysfunction. Transfection of human lung microvascular ECs with paxillin Y31, Y118 and Y31/Y118 mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization at adherens junctions. In vivo, knockdown of paxillin with siRNA in mouse lungs ameliorated LPS-induced pulmonary protein leak and lung inflammation. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

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Tests by Tissue Culture Methods on the Nature of Immunity Transplanted Skin

Journal of Cell Science ◽

10.1242/jcs.s3-89.7.239 ◽

1948 ◽

Vol s3-89 (7) ◽

pp. 239-252

Author(s):

P. B. MEDAWAR

Keyword(s):

Tissue Culture ◽

Cell Division ◽

Epidermal Cell ◽

Cell Suspensions ◽

Acquired Immunity ◽

Culture Methods ◽

Division Frequency

The transplantation of skin from one rabbit to another elicits a reaction that conforms in main outline with that of an actively acquired immunity. The experiments described in this paper were designed to test the hypothesis that the regression of such grafts is secured by the action of antibodies demonstrable in vitro. Skin from adult rabbits has therefore been cultivated in the presence of serum and growing mesenchymal tissues derived solely from rabbits heavily and specifically immunized against it. Immune sera and tissues are without effect on the survival, cell-division frequency and migratory activities of explanted skin, and agglutinins for epidermal cell suspensions are not demonstrable in immune sera. With certain stated qualifications, it has therefore been concluded that the occurrence of free antibodies is not a sufficient explanation of the regression of skin homografts in vivo.

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