scholarly journals Src-dependent NM2A tyrosine-phosphorylation regulates actomyosin dynamics

2020 ◽  
Author(s):  
Cláudia Brito ◽  
Francisco S. Mesquita ◽  
Daniel S. Osório ◽  
Joana Pereira ◽  
Neil Billington ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Biology ◽  
Bacterial Infections ◽  
Focal Adhesions ◽  
The Status ◽  
Fine Control ◽  
Actomyosin Cytoskeleton

AbstractNon-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that along with actin assembles into actomyosin filaments inside cells. NM2A is fundamental in cellular processes requiring force generation such as cell adhesion, motility and cell division, and plays important functions in different stages of development and during the progression of viral and bacterial infections. We previously identified at the motor domain of the NM2A, a novel Src-dependent tyrosine phosphorylation on residue 158 (pTyr158), which is promoted by Listeria monocytogenes infection. Despite the central role of NM2A in several cell biology processes, the pTyr at this specific residue had never been reported. Here we showed that LLO, a toxin secreted by Listeria, is sufficient to trigger NM2A pTyr158 by activating Src, which coordinates actomyosin remodeling. We further addressed the role of NM2A pTyr158 on the organization and dynamics of the actomyosin cytoskeleton and found that by controlling the activation of the NM2A, the status of the pTyr158 alters cytoskeletal organization, dynamics of focal adhesions and cell motility, without affecting NM2A enzymatic activity in vitro. Ultimately, by using Caenorhabditis elegans as a model to assess the role of this pTyr158in vivo, we found that the status of the pTyr158 has implications in gonad function and is required for organism survival under stress conditions. We conclude that the fine control of the NM2A pTyr158 is required for cell cytoskeletal remodeling and dynamics, and we propose Src-dependent NM2A pTyr158 as a novel layer of regulation of the actomyosin cytoskeleton.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

2007 ◽  
Vol 204 (13) ◽  
pp. 3103-3111 ◽  
Author(s):  
Brian G. Petrich ◽  
Patrizia Marchese ◽  
Zaverio M. Ruggeri ◽  
Saskia Spiess ◽  
Rachel A.M. Weichert ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Ex Vivo ◽  
Focal Adhesions ◽  
Β1 Integrin ◽  
Normal Morphology ◽  
And Function ◽  
Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 95-95 ◽  
Author(s):  
Keisuke Ito ◽  
Paolo Sportoletti ◽  
John G Clohessy ◽  
Grisendi Silvia ◽  
Pier Paolo Pandolfi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Cell Biology ◽  
De Novo ◽  
Stem Cell Biology ◽  
Hematopoietic Stem

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

β3 Tyrosine Phosphorylation in αIIbβ3 (Platelet Membrane GP IIb-IIIa) Outside-in Integrin Signaling

2001 ◽  
Vol 86 (07) ◽  
pp. 246-258 ◽  
Author(s):  
Lisa Nannizzi-Alaimo ◽  
K. S. Srinivasa Prasad ◽  
David Phillips
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Critical Role ◽  
Clot Retraction ◽  
Signaling Mechanism ◽  
Platelet Aggregate ◽  
Von Willebrand

SummaryThe platelet integrin αIIbβ3 not only binds fibrinogen and von Willebrand factor to mediate platelet aggregation and adhesion, it also serves as a signaling receptor. Platelet agonists such as ADP, thrombin and collagen induce “inside-out” signaling which activates the receptor function of αIIbβ3 for soluble fibrinogen. Subsequent platelet aggregation leads to “outside-in” signaling, inducing platelet aggregate stabilization and triggering a variety of functions important to platelet physiology. This review focuses on the role of β3 tyrosine phosphorylation in αIIbβ3 outside-in signaling. Tyrosine phosphorylation of β3 in platelets is a dynamic process which is initiated upon platelet aggregation and also by adhesion of platelets to immobilized fibrinogen. Tyrosine phosphorylation occurs on the β3 integrin cytoplasmic tyrosine (ICY) domain, a conserved motif found in thesubunits of several integrins. β3 ICY domain tyrosine phosphorylation induces the recruitment of two proteins to the cytoplasmic domains of αIIbβ3: the cytoskeletal protein myosin, important to clot retraction; and the signaling adapter protein Shc, important to platelet stimulation. The critical role of β3 tyrosine phosphorylation to platelet function was established by the diYF mouse, a novel strain which expresses an αIIbβ3 in which the two β3 ICY domain tyrosines have been mutated to phenylalanine. These mice are selectively impaired in outside-in αIIbβ3 signaling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. Taken together, the data suggest that the β3 tyrosine phosphorylation signaling mechanism is important to αIIbβ3 function and might be applicable to a wide variety of integrin-mediated events.

scholarly journals Iron, copper and immunocompetence

2007 ◽  
Vol 98 (S1) ◽  
pp. S24-S28 ◽  
Author(s):  
Carlos Muñoz ◽  
Ernesto Rios ◽  
Jorge Olivos ◽  
Oscar Brunser ◽  
Manuel Olivares
Keyword(s):  
Cell Biology ◽  
Respiratory Infections ◽  
The Elderly ◽  
Iron Nutrition ◽  
Immune Functions ◽  
Human Beings ◽  
Additional Information

Microminerals including copper and iron are essential to immunity and health in human beings. The development of powerful tools in analytical cell biology and molecular genetics has facilitated efforts to identify specific cellular and molecular functions of trace elements in the maturation, activation and functions of host defence mechanisms. Selected recent reports about the role of copper and iron nutrition on immune functions are critically analysed here. Effects of trace element supplementation on infectious morbidity are also reviewed. While micromineral deficiencies, in general, may have widespread effects on nearly all components of immune response, these effects can be reversed by supplementation. However, the conflicting effects of iron deficiency and iron supplementationin vitroon the defensive systems reveals the urgent need for further additional information on thein vivosituation. In the elderly, vaccination against respiratory infections is likely to protect only 30–70 % of the population. However, it may be possible to modulate immune function and ultimately reduce the severity of infections through micronutrient supplementation. Thus, microminerals contribute to the maintenance of the balance between immunity and health in humans.

scholarly journals Molecular mechanism and physiological role of active–deactive transition of mitochondrial complex I

2013 ◽  
Vol 41 (5) ◽  
pp. 1325-1330 ◽  
Author(s):  
Marion Babot ◽  
Alexander Galkin
Keyword(s):  
Complex I ◽  
Physiological Role ◽  
Protective Mechanism ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
The Status ◽  
Nitric Oxide Metabolites

The unique feature of mitochondrial complex I is the so-called A/D transition (active–deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~104 min−1) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1–10 min−1) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys39 of mitochondrially encoded subunit ND3 makes the D-form susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.

miR-223 regulates proliferation and apoptosis by targeting insulin-like growth factor 1 receptor (IGF1R) in osteosarcoma cells

2019 ◽  
Vol 9 (5) ◽  
pp. 459-466
Author(s):  
Jianhua Xiao ◽  
Chunlei He ◽  
Yanchun Zhong ◽  
Lulin Liu ◽  
Yaosheng Xiao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cancer Progression ◽  
Tumor Suppressor Genes ◽  
Cell Biology ◽  
Cell Activity ◽  
Insulin Like Growth Factor ◽  
Proliferation And Apoptosis

MicroRNAs are widely involved in cancer progression by inhibiting the expression levels of oncogenes or tumor-suppressor genes, and the dysregulation of microRNAs may contribute to tumorigenesis. Here, we use molecular-biology and cell-biology methods to determine the role of miR-223 in osteosarcoma (OS) regulation. The regulating effect of miR-223 on OS is determined by various experimental methods in vivo and in vitro. Exogenous miR-223 reduces the proliferation of HOS and MG-63 cells in vitro. In addition, we show that excessive expression of miR-223 significantly increases apoptosis of OS cells. Moreover, the insulin-like growth factor 1 receptor (IGF-1R) is downgraded after using an miR-223 simulated transfection in HOS and MG-63 cells. After IGF-1R inhibition, the cell activity of the HOS and MG-63 cells is also inhibited. In addition, we demonstrate that miR-223 inhibits OS proliferation and induces apoptosis by targeting a point in IGF-1R 3′ -UTR. In summary, miR-223 inhibits OS proliferation and induces apoptosis by targeting IGF-1R. New therapies with fewer complications based on miRNAs should be studied.

scholarly journals Human Enteric Defensin 5 Promotes Shigella Infection of Macrophages

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Dan Xu ◽  
Chongbing Liao ◽  
Jiu Xiao ◽  
Kun Fang ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Protective Factor ◽  
Antimicrobial Activities ◽  
The Status ◽  
Host Defense Peptide ◽  
Type 3 Secretion System ◽  
Type 3

ABSTRACT Human α-defensins are 3- to 5-kDa disulfide-bridged peptides with a multitude of antimicrobial activities and immunomodulatory functions. Recent studies show that human enteric α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, aids the highly infectious enteropathogen Shigella in breaching the intestinal epithelium in vitro and in vivo. Whether and how HD5 influences Shigella infection of resident macrophages following its invasion of the intestinal epithelium remain poorly understood. Here, we report that HD5 greatly promoted phagocytosis of Shigella by macrophages by targeting the bacteria to enhance bacterium-to-cell contacts in a structure- and sequence-dependent fashion. Subsequent intracellular multiplication of phagocytosed Shigella led to massive necrotic cell death and release of the bacteria. HD5-promoted phagocytosis of Shigella was independent of the status of the type 3 secretion system. Furthermore, HD5 neither inhibited nor enhanced phagosomal escape of Shigella. Collectively, these findings confirm a potential pathogenic role of HD5 in Shigella infection of not only epithelial cells but also macrophages, illuminating how an enteropathogen exploits a host protective factor for virulence and infection.

Effects of cadmium on the actin cytoskeleton in renal mesangial cells

2013 ◽  
Vol 91 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Douglas M. Templeton ◽  
Ying Liu
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Mesangial Cells ◽  
Focal Adhesions ◽  
Filamentous Actin ◽  
Human Mesangial Cells ◽  
Dependent Protein

We provide an overview of our studies on cadmium and the actin cytoskeleton in mesangial cells, from earlier work on the effects of Cd2+ on actin polymerization in vivo and in vitro, to a role of disruption or stabilization of the cytoskeleton in apoptosis and apoptosis-like death. More recent studies implicate cadmium-dependent association of gelsolin and the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with actin filaments in cytoskeletal effects. We also present previously unpublished data concerning cadmium and the disruption of focal adhesions. The work encompasses studies on rat, mouse, and human mesangial cells. The major conclusions are that Cd2+ acts independently of direct effects on cellular Ca2+ levels to nevertheless activate Ca2+-dependent proteins that shift the actin polymerization–depolymerization in favour of depolymerization. Cadmium-dependent translocation of CaMK-IIδ, gelsolin, and a 50 kDa gelsolin cleavage fragment to the filamentous (F-)actin cytoskeleton appear to be involved. An intact filamentous actin cytoskeleton is required to initiate apoptotic and apoptotic-like death, but F-actin depolymerization is an eventual result.

scholarly journals The Role of Candida albicans NOT5 in Virulence Depends upon Diverse Host Factors In Vivo

2005 ◽  
Vol 73 (11) ◽  
pp. 7190-7197 ◽  
Author(s):  
Shaoji Cheng ◽  
Cornelius J. Clancy ◽  
Mary Ann Checkley ◽  
Zongde Zhang ◽  
Karen L. Wozniak ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Functional Equivalence ◽  
Host Defenses ◽  
Growth Defects ◽  
The Status ◽  
Portal Of Entry ◽  
Tissue Site

ABSTRACT We previously identified Candida albicans Not5p as an immunogenic protein expressed during oropharyngeal candidiasis (OPC). In this study, we demonstrate that C. albicans NOT5 reverses the growth defects of a Saccharomyces cerevisiae not5 mutant strain at 37°C, suggesting that the genes share at least some functional equivalence. We implicate C. albicans NOT5 in the pathogenesis of disseminated candidiasis (DC) induced by intravenous infection among neutropenic and nonimmunosuppressed mice, as well as in that of OPC in mice immunosuppressed with corticosteroids. We find no role in virulence, however, among neutropenic and corticosteroid-suppressed mice with DC resulting from gastrointestinal translocation, nor do we implicate the gene in vulvovaginal candidiasis among mice in pseudoestrus. These findings suggest that the role of NOT5 in virulence depends on the specific in vivo environment and is influenced by diverse factors such as tissue site, portal of entry, and the status of host defenses. NOT5 is necessary for normal adherence to colonic and cervical epithelial cells in vitro, demonstrating that such assays cannot fully replicate disease processes in vivo. Lastly, antibody responses against Not5p do not differ in the sera of patients with OPC, patients with DC, and healthy controls, suggesting that the protein is associated with both commensalism and the pathogenesis of disease.

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