Toxoplasma gondii establishes an extensive filamentous network consisting of stable F-actin during replication

AbstractApicomplexan actin is well conserved and clearly important during the parasite's life cycle. Several studies assert that its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. However, it has not been possible to study actin in vivo, so its physiological role has remained obscure. This has led to functional models which are mutually conflicting, incompatible with actin behavior from other eukaryotes, and cannot explain actin's importance during basic processes such as parasite replication and egress. Here we use a chromobody that specifically binds F-actin to demonstrate that Toxoplasma forms stable actin filaments in vivo. F-actin is not only important for parasite replication, but also forms an extensive network that connects individuals both within and between parasitophorous vacuoles, and allows vesicles to be exchanged between parasites within a vacuole. During host cell egress, prior to motility, this network collapses in a calcium-dependent manner. This study demonstrates unexpected roles of Toxoplasma actin during the asexual life cycle, and proves that formation of F-actin depends on a critical concentration of G-actin, implying a polymerization mechanism similar to mammalian actin.One Sentence SummaryToxoplasma establishes a stable F-actin network that is essential for replication and material transport between individual parasites.

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Increased Bone Mass in Mice Lacking the Adipokine Apelin

Endocrinology ◽

10.1210/en.2012-2034 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2069-2080 ◽

Cited By ~ 22

Author(s):

Lalita Wattanachanya ◽

Wei-Dar Lu ◽

Ramendra K. Kundu ◽

Liping Wang ◽

Marcia J. Abbott ◽

...

Keyword(s):

Bone Mass ◽

Bone Cells ◽

Physiological Role ◽

In Vitro Study ◽

Maximum Effect ◽

Dependent Manner ◽

Primary Mouse ◽

Skeletal Homeostasis

Abstract Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

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Reticulon 3-mediated Chk2/p53 activation suppresses hepatocellular carcinogenesis and is blocked by hepatitis B virus

Gut ◽

10.1136/gutjnl-2020-321386 ◽

2020 ◽

pp. gutjnl-2020-321386

Author(s):

Shushu Song ◽

Yinghong Shi ◽

Weicheng Wu ◽

Hao Wu ◽

Lei Chang ◽

...

Keyword(s):

Cellular Proliferation ◽

Dependent Manner ◽

Mice Model ◽

Calcium Dependent ◽

B Virus ◽

P53 Activation ◽

Underlying Mechanisms ◽

Induced Apoptosis

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms.DesignClinical HCC samples were collected to assess the relationship between RTN3 expression and patients’ outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo.ResultsWe found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation.ConclusionThe findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.

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β-Chemokines Enhance Parasite Uptake and Promote Nitric Oxide-Dependent Microbiostatic Activity in Murine Inflammatory Macrophages Infected with Trypanosoma cruzi

Infection and Immunity ◽

10.1128/iai.67.9.4819-4826.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4819-4826 ◽

Cited By ~ 101

Author(s):

Júlio C. S. Aliberti ◽

Fabiana S. Machado ◽

Janeusa T. Souto ◽

Ana P. Campanelli ◽

Mauro M. Teixeira ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Neutralizing Antibodies ◽

Specific Inhibitor ◽

No Production ◽

Dependent Manner ◽

Macrophage Infection ◽

Parasite Replication ◽

Ifn Γ

ABSTRACT In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of β-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1α, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of β-chemokine MIP-1α, MIP-1β, RANTES, and JE mRNA by macrophages. The expression of the β-chemokine MIP-1α, MIP-1β, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms ofT. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-β-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous β-chemokines MIP-1α, MIP-1β, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. l-NMMA, a specific inhibitor of thel-arginine–NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with β-chemokines. Among the β-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-γ). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruziinfection.

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Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells

Blood ◽

10.1182/blood.v67.5.1281.bloodjournal6751281 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1281-1285

Author(s):

EA Thompson ◽

MA Howard

Keyword(s):

Von Willebrand Factor ◽

Cell Activation ◽

Calcium Ionophore ◽

Ionophore A23187 ◽

Polymorphonuclear Cells ◽

Dependent Manner ◽

Calcium Dependent ◽

Von Willebrand ◽

Willebrand Factor

In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio- CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.

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Annexin VI: an intracellular target for ATP.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4152 ◽

1999 ◽

Vol 46 (3) ◽

pp. 801-812 ◽

Cited By ~ 3

Author(s):

J Bandorowicz-Pikuła ◽

M Danieluk ◽

A Wrzosek ◽

R Buś ◽

R Buchet ◽

...

Keyword(s):

Nucleotide Binding ◽

Dependent Manner ◽

Amino Acid Residues ◽

Binding Motifs ◽

Calcium Dependent ◽

Annexin Vi ◽

Self Association ◽

Nucleotide Binding Proteins

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.

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An Exaggerated Immune Response in Female BALB/c Mice Controls Initial Toxoplasma gondii Multiplication but Increases Mortality and Morbidity Relative to Male Mice

Pathogens ◽

10.3390/pathogens10091154 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1154

Author(s):

Rasha Alonaizan ◽

Stuart Woods ◽

Kerrie E Hargrave ◽

Craig W. Roberts

Keyword(s):

Immune Response ◽

Imaging System ◽

Acute Infection ◽

Mortality Rates ◽

Dependent Manner ◽

Male Mice ◽

Female Mice ◽

Parasite Multiplication ◽

Parasite Replication

Studies indicate that female mice are more susceptible to T. gondii infection, as defined by higher mortality rates in comparison to male mice. However, whether this is due to an inability to control initial parasite multiplication or due to detrimental effects of the immune system has not been determined. Therefore, the following studies were undertaken to determine the influence of sex on early parasite multiplication and the immune response during T. gondii infection and to correlate this with disease outcome. Early parasite replication was studied through applying an in vivo imaging system (IVIS) with luciferase expressing T. gondii. In parallel immunological events were studied by cytometric bead array to quantify key immunological mediators. The results confirmed the previous findings that female mice are more susceptible to acute infection, as determined by higher mortality rates and weight loss compared with males. However, conflicting with expectations, female mice had lower parasite burdens during the acute infection than male mice. Female mice also exhibited significantly increased production of Monocyte Chemoattractant Protein-1 (MCP-1), Interferon (IFN)-γ, and Tumour Necrosis Factor (TNF)-α than male mice. MCP-1 was found to be induced by T. gondii in a dose dependent manner suggesting that the observed increased levels detected in female mice was due to a host-mediated sex difference rather than due to parasite load. However, MCP-1 was not affected by physiological concentration of estrogen or testosterone, indicating that MCP-1 differences observed between the sexes in vivo are due to an as yet unidentified intermediary factor that in turn influences MCP-1 levels. These results suggest that a stronger immune response in female mice compared with male mice enhances their ability to control parasite replication but increases their morbidity and mortality.

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The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments

Journal of Cell Science ◽

10.1242/jcs.112.22.3943 ◽

1999 ◽

Vol 112 (22) ◽

pp. 3943-3954 ◽

Cited By ~ 1

Author(s):

V. Assmann ◽

D. Jenkinson ◽

J.F. Marshall ◽

I.R. Hart

Keyword(s):

Actin Filaments ◽

Human Cancer ◽

Cell Surface Receptor ◽

Dependent Manner ◽

Interaction Domain ◽

Microtubule Binding ◽

Projection Domain ◽

Hyaluronan Receptor

We reported recently on the intracellular localisation of the hyaluronan receptor RHAMM/IHABP in human cancer cells. Here we describe the colocalisation of RHAMM/IHABP proteins with microtubules, both in interphase and dividing cells, suggesting that RHAMM/IHABP represents a novel member of the family of microtubule-associated proteins (MAPs). We have identified four different splice variants of RHAMM/IHABP, all of which colocalise, at least transiently, with microtubules when expressed as GFP fusion proteins in HeLa cells. Using microtubule-binding assays and transient transfection experiments of deletion-bearing RHAMM/IHABP mutants, we localised the microtubule-binding region to the extreme N terminus of RHAMM/IHABP. This interaction domain is composed of two distinct subdomains, one of which is sufficient to mediate binding to the mitotic spindle while both domains are required for binding of RHAMM/IHABP proteins to interphase microtubules. Sequence analysis revealed that the projection domain of RHAMM/IHABP is predicted to form coiled-coils, implying that RHAMM/IHABP represents a filamentous protein capable of interacting with other proteins and we found that RHAMM/IHABP interacts with actin filaments in vivo and in vitro. Moreover, in vitro translated RHAMM/IHABP isoforms efficiently bind to immobilised calmodulin in a Ca(2+)-dependent manner via a calmodulin-binding site within the projection domain of RHAMM/IHABP (residues 574–602). Taken together, our results strongly suggest that RHAMM/IHABP is a ubiquitously expressed, filamentous protein capable of interacting with microtubules and microfilaments and not, as numerous previous reports suggest, a cell surface receptor for the extracellular matrix component hyaluronan.

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Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function

10.1101/2020.12.30.424812 ◽

2021 ◽

Author(s):

Asma B. Salek ◽

Ruchi Bansal ◽

Nicolas F. Berbari ◽

Anthony J. Baucum

Keyword(s):

Cognitive Flexibility ◽

Hippocampal Neurons ◽

Calcium Influx ◽

Surface Expression ◽

List Type ◽

Dependent Manner ◽

Caspase Cleavage ◽

Calcium Dependent ◽

Apoptotic Pathways

ABSTRACTN-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction.HIGHLIGHTSSpinophilin bidirectionally regulates GluN2B-containing NMDAR function.Loss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker.Loss of spinophilin in vivo decreases measures of spatial cognitive flexibility.Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks.

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How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper.

The Journal of Cell Biology ◽

10.1083/jcb.118.1.83 ◽

1992 ◽

Vol 118 (1) ◽

pp. 83-93 ◽

Cited By ~ 65

Author(s):

L G Tilney ◽

D J DeRosier ◽

A Weber ◽

M S Tilney

Keyword(s):

Host Cell ◽

Actin Filaments ◽

Critical Concentration ◽

Filament Assembly ◽

Tightly Coupled ◽

Pointed End ◽

Phagosomal Membrane ◽

Simple Picture

After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.

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The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.45 ◽

1996 ◽

Vol 16 (1) ◽

pp. 45-52 ◽

Cited By ~ 81

Author(s):

V Ribon ◽

S Hubbell ◽

R Herrera ◽

A R Saltiel

Keyword(s):

Tyrosine Phosphorylation ◽

Leukemia Cell ◽

Physiological Role ◽

Sh2 Domain ◽

Myelogenous Leukemia ◽

Leukemia Cell Line ◽

Stable Complex ◽

Binding Motif ◽

Dependent Manner

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.

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