scholarly journals TRPV2 activation reorganizes actin cytoskeleton, induces neurite initiation and branching by altering cAMP levels

2019 ◽  
Author(s):  
Manoj Yadav ◽  
Chandan Goswami
Keyword(s):  
Actin Cytoskeleton ◽  
Molecular Level ◽  
Ectopic Expression ◽  
Wild Type ◽  
Filamentous Actin ◽  
Peripheral Neuron ◽  
Neurite Initiation ◽  
Intracellular Ca ◽  
Camp Levels

AbstractUnderstanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important by which neuronal damages can be repaired. In this work we explored the role of TRPV2, a non-selective cation channel in the context of neurite functions. Using western blot, immunofluorescence, and live cell Ca2+-imaging; we confirm that functional TRPV2 is endogenously present in the F11 cell, a model system mimicking peripheral neuron. In F11 cells TRPV2 localizes in specific sub-cellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP but not GFP-only in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wild-type TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. However, TRPV2-mediated neuritogenesis is unperturbed by the chelation of intracellular Ca2+ by BAPTA-AM, and thus involves Ca2+-independent signaling events also. We demonstrate that pharmacological modulation of TRPV2 alters cellular cAMP levels. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular level. Such understanding may have border implication in neurodegeneration and peripheral neuropathy.

scholarly journals TRPV2 interacts with actin and reorganizes submembranous actin cytoskeleton

2020 ◽  
Vol 40 (10) ◽  
Author(s):  
Manoj Yadav ◽  
Chandan Goswami
Keyword(s):  
Actin Cytoskeleton ◽  
Transient Receptor Potential ◽  
Neuronal Damage ◽  
Ectopic Expression ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Filamentous Actin ◽  
Peripheral Neuron ◽  
Neurite Initiation ◽  
Transient Receptor

Abstract The understanding of molecules and their role in neurite initiation and/or extension is not only helpful to prevent different neurodegenerative diseases but also can be important in neuronal damage repair. In this work, we explored the role of transient receptor potential vanilloid 2 (TRPV2), a non-selective cation channel in the context of neurite functions. We confirm that functional TRPV2 is endogenously present in F11 cell line, a model system mimicking peripheral neuron. In F11 cells, TRPV2 localizes in specific subcellular regions enriched with filamentous actin, such as in growth cone, filopodia, lamellipodia and in neurites. TRPV2 regulates actin cytoskeleton and also interacts with soluble actin. Ectopic expression of TRPV2-GFP in F11 cell induces more primary and secondary neurites, confirming its role in neurite initiation, extension and branching events. TRPV2-mediated neuritogenesis is dependent on wildtype TRPV2 as cells expressing TRPV2 mutants reveal no neuritogenesis. These findings are relevant to understand the sprouting of new neurites, neuroregeneration and neuronal plasticity at the cellular, subcellular and molecular levels. Such understanding may have further implications in neurodegeneration and peripheral neuropathy.

Role of Lipid Rafts and the Underlying Filamentous-Actin Cytoskeleton in Cannabinoid Receptor 1 Signaling

2016 ◽  
pp. 689-701
Author(s):  
Dimitra Mangoura ◽  
Olga Asimaki ◽  
Emmanouella Tsirimonaki ◽  
Nikos Sakellaridis
Keyword(s):  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
Cannabinoid Receptor ◽  
Filamentous Actin ◽  
Cannabinoid Receptor 1

scholarly journals Loss of Arc attenuates the behavioral and molecular responses for sleep homeostasis in mice

2020 ◽  
Vol 117 (19) ◽  
pp. 10547-10553 ◽  
Author(s):  
Ayako Suzuki ◽  
Masashi Yanagisawa ◽  
Robert W. Greene
Keyword(s):  
Messenger Rna ◽  
Potential Role ◽  
Molecular Level ◽  
Subcellular Location ◽  
Early Gene ◽  
Wild Type ◽  
Molecular Responses ◽  
Recovery Sleep ◽  
Sleep Homeostasis

The activity-regulated cytoskeleton-associated protein (Arc) gene is a neural immediate early gene that is involved in synaptic downscaling and is robustly induced by prolonged wakefulness in rodent brains. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strengthening. This suggests a potential role for Arc in these and other sleep-related processes. However, the role of Arc in sleep remains unknown. Here, we demonstrated that Arc is important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis. Arc knockout (KO) mice displayed increased time spent in rapid eye movement (REM) sleep under baseline conditions and marked attenuation of sleep rebound to both 4 h of total sleep deprivation (SD) and selective REM deprivation. At the molecular level, the following homeostatic sleep responses to 4-h SD were all blunted in Arc KO mice: increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 and its phosphorylation in synaptoneurosomes; induction of a subset of SD-response genes; and suppression of the GluA1 messenger RNA in the cortex. In wild-type brains, SD increased Arc protein expression in multiple subcellular locations, including the nucleus, cytoplasm, and synapse, which is reversed in part by recovery sleep. Arc is critical for these behavioral and multiple molecular responses to SD, thus providing a multifunctional role for Arc in the maintenance of sleep homeostasis, which may be attributed by the sleep/wake-associated changes in subcellular location of Arc.

scholarly journals Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA

2014 ◽  
Vol 27 (7) ◽  
pp. 603-610 ◽  
Author(s):  
Michiel J. C. Pel ◽  
Anja J. H. van Dijken ◽  
Bart W. Bardoel ◽  
Michael F. Seidl ◽  
Sjoerd van der Ent ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Alkaline Protease ◽  
Bacterial Species ◽  
Gene Activation ◽  
Ectopic Expression ◽  
Host Immunity ◽  
Wild Type ◽  
Knockout Mutant ◽  
Inhibitory Peptide

Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

scholarly journals Role of the H subunit C-terminal domain in the assembly of the vacuolar H+-ATPase

2018 ◽  
Author(s):  
Stuti Sharma ◽  
Rebecca A. Oot ◽  
Stephan Wilkens
Keyword(s):  
Biochemical Analysis ◽  
Molecular Level ◽  
Model Organism ◽  
Proton Channel ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
C Terminus ◽  
Rotary Motor

AbstractThe vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor and recent structural analysis revealed that disassembled V1 and Vo are in different rotary states, a mismatch that is likely responsible for the inability to reconstitute holo V-ATPase from its functional sectors in vitro. Here, using the model organism S. cerevisiae, we show that a key impediment for binding of autoinhibited V1 to Vo is the conformation of the inhibitory C-terminus of subunit H (HCT). Using biolayer interferometry and biochemical analysis, we show that selective disruption of HCT’s binding site on V1 allows in vitro assembly of a structurally and functionally coupled V-ATPase complex. The resultant mutant V-ATPase, however, does not disassemble as readily as the wild type enzyme, highlighting the importance of HCT’s conformation in the mechanism of reversible disassembly. These findings pave the way for identifying molecules that allow for therapeutic modulation of aberrant V-ATPase activity in the disease state.

VAMP-7 Interacts With The Actin Cytoskeleton To Control Platelet Spreading and Mediate α-Granule Exocytosis During Thrombus Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1058-1058
Author(s):  
Secil Koseoglu ◽  
Jennifer L Fitch-Tewfik ◽  
Christian G. Peters ◽  
Lydia Danglot ◽  
Thierry Galli ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Wild Type ◽  
Granule Exocytosis ◽  
Granule Secretion ◽  
Platelet Spreading

Abstract Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7+ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice were used to analyze the role of VAMP-7 in platelet spreading, aggregation and secretion. VAMP-7 null platelets were normal in size, shape, and number. When compared to wild-type platelets, VAMP-7 null platelets did not show any defects in aggregation upon exposure to increasing doses of the PAR4 agonist peptide, AYPGKF, or collagen. In contrast, the surface area of VAMP-7 null platelets following 15 min of spreading on poly-L-lysine was only 51% that of wild-type of platelets (P < 0.05). To assess mechanisms of the movement of VAMP-7 to the platelet periphery, the association of VAMP-7 to the Triton X-100-insoluble platelet cytoskeleton was evaluated and results showed that VAMP-7 associated with the actin cytoskeleton. Moreover, VAMP-7 null platelets showed impaired P-selectin surface expression and PF4 secretion at low concentrations of AYPGKF. TIMP-2 and VEGF localize to VAMP-7 expressing granules in the periphery of spread platelets. We therefore evaluated the secretion of TIMP-2 and VEGF from VAMP-7 null platelets. Secretion of TIMP-2 and VEGF was reduced even at saturating doses of agonist (300 mM AYPGKF). To examine the role of VAMP-7 in a-granule exocytosis during platelet activation in vivo, PF4 release was monitored following laser-induced injury of cremaster arterioles. Platelet accumulation at sites of laser injury was identical in wild-type and VAMP-7 null mice. In wild-type mice, PF4 was secreted by activated platelets and bound back to activated endothelium and platelets producing a localized concentration of PF4 that accumulated over 15 min following injury. PF4 release from platelets lacking VAMP-7 was decreased to 47% of that of control. These results demonstrate that VAMP-7 interacts with the actin cytoskeleton and functions selectively in a-granule exocytosis. VAMP-7 associates with the actin cytoskeleton and functions during platelet spreading, adding further support to the premise that membrane fusion occurring during granule secretion is an essential component of normal platelet spreading. This VAMP-7 mediated, actin-dependent mechanism of secretion is not important for platelet thrombus formation, but rather functions in the release of particular granular contents, such as PF4, at sites of vascular injury. Disclosures: No relevant conflicts of interest to declare.

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 Lung Epithelial TRPA1 Transduces the Extracellular ROS into Transcriptional Regulation of Lung Inflammation Induced by Cigarette Smoke: The Role of Influxed Ca2+

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
An-Hsuan Lin ◽  
Meng-Han Liu ◽  
Hsin-Kuo Ko ◽  
Diahn-Warng Perng ◽  
Tzong-Shyuan Lee ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Cigarette Smoke ◽  
Lung Inflammation ◽  
Lung Epithelial Cells ◽  
Wild Type ◽  
Intracellular Ros ◽  
Lung Epithelial ◽  
Intracellular Ca ◽  
Acetyl Cysteine

The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca2+influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca2+with the chelator EGTA, or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA, or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine, or HC030031. The activation of the MAPKs/NF-κB signaling was suppressed by EGTA, N-acetyl-cysteine, or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposuretrpa1−/−mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice; yet lung inflammation was lessened. Thus, in LECs, CSE may initially increase extracellular ROS, which activate TRPA1 leading to an increase in Ca2+influx. The increased intracellular Ca2+contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/NF-κB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.

scholarly journals CaV3.2 T-type Ca2+ channels in H2S-mediated hypoxic response of the carotid body

2015 ◽  
Vol 308 (2) ◽  
pp. C146-C154 ◽  
Author(s):  
Vladislav V. Makarenko ◽  
Ying-Jie Peng ◽  
Guoxiang Yuan ◽  
Aaron P. Fox ◽  
Ganesh K. Kumar ◽  
...  
Keyword(s):  
Carotid Body ◽  
Sensory Nerve ◽  
Wild Type ◽  
Glomus Cells ◽  
Arterial Blood ◽  
Carotid Bodies ◽  
Intracellular Ca ◽  
Ca2 Channels ◽  
Body Response

Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca2+ channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca2+ concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout ( Cacna1h−/−) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca2+ concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h−/− mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia.

scholarly journals Role of GerD in Germination of Bacillus subtilis Spores

2006 ◽  
Vol 189 (3) ◽  
pp. 1090-1098 ◽  
Author(s):  
Patricia L. Pelczar ◽  
Takao Igarashi ◽  
Barbara Setlow ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Cysteine Residue ◽  
Wild Type ◽  
Hypertonic Medium ◽  
Putative Signal Peptide

ABSTRACT Spores of a Bacillus subtilis strain with a gerD deletion mutation (ΔgerD) responded much slower than wild-type spores to nutrient germinants, although they did ultimately germinate, outgrow, and form colonies. Spores lacking GerD and nutrient germinant receptors also germinated slowly with nutrients, as did ΔgerD spores in which nutrient receptors were overexpressed. The germination defect of ΔgerD spores was not suppressed by many changes in the sporulation or germination conditions. Germination of ΔgerD spores was also slower than that of wild-type spores with a pressure of 150 MPa, which triggers spore germination through nutrient receptors. Ectopic expression of gerD suppressed the slow germination of ΔgerD spores with nutrients, but overexpression of GerD did not increase rates of spore germination. Loss of GerD had no effect on spore germination induced by agents that do not act through nutrient receptors, including a 1:1 chelate of Ca2+ and dipicolinic acid, dodecylamine, lysozyme in hypertonic medium, a pressure of 500 MPa, and spontaneous germination of spores that lack all nutrient receptors. Deletion of GerD's putative signal peptide or change of its likely diacylglycerylated cysteine residue to alanine reduced GerD function. The latter findings suggest that GerD is located in a spore membrane, most likely the inner membrane, where the nutrient receptors are located. All these data suggest that, while GerD is not essential for nutrient germination, this protein has an important role in spores' rapid response to nutrient germinants, by either direct interaction with nutrient receptors or some signal transduction essential for germination.

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