Sequential phosphorylation of NDEL1 by the DYRK2-GSK3β complex is critical for neuronal morphogenesis

Neuronal morphogenesis requires multiple regulatory pathways to appropriately determine axonal and dendritic structures, thereby to enable the functional neural connectivity. Yet, however, the precise mechanisms and components that regulate neuronal morphogenesis are still largely unknown. Here, we newly identified the sequential phosphorylation of NDEL1 critical for neuronal morphogenesis through the human kinome screening and phospho-proteomics analysis of NDEL1 from mouse brain lysate. DYRK2 phosphorylates NDEL1 S336 to prime the phosphorylation of NDEL1 S332 by GSK3β. TARA, an interaction partner of NDEL1, scaffolds DYRK2 and GSK3β to form a tripartite complex and enhances NDEL1 S336/S332 phosphorylation. This dual phosphorylation increases the filamentous actin dynamics. Ultimately, the phosphorylation enhances both axonal and dendritic outgrowth and promotes their arborization. Together, our findings suggest the NDEL1 phosphorylation at S336/S332 by the TARA-DYRK2-GSK3β complex as a novel regulatory mechanism underlying neuronal morphogenesis.

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Effect of destrin mutations on the gene expression profile in vivo

Physiological Genomics ◽

10.1152/physiolgenomics.00285.2007 ◽

2008 ◽

Vol 34 (1) ◽

pp. 9-21 ◽

Cited By ~ 24

Author(s):

Angela M. Verdoni ◽

Natsuyo Aoyama ◽

Akihiro Ikeda ◽

Sakae Ikeda

Keyword(s):

Gene Expression ◽

Gene Expression Profile ◽

Expression Profile ◽

Target Genes ◽

Actin Dynamics ◽

In Vivo Model ◽

Strong Impact ◽

Filamentous Actin ◽

Control Of Gene Expression

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 ( corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn corn1 mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn corn1 mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn corn1 and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn corn1 cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

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Proline-rich transmembrane protein 2 (PRRT2) regulates the actin cytoskeleton during synaptogenesis

Cell Death and Disease ◽

10.1038/s41419-020-03073-w ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Elisa Savino ◽

Romina Inès Cervigni ◽

Miriana Povolo ◽

Alessandra Stefanetti ◽

Daniele Ferrante ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Neurotransmitter Release ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Transmembrane Protein ◽

Neuronal Cell ◽

Cell Aggregation ◽

Actin Dynamics ◽

Actin Binding ◽

Filamentous Actin

Abstract Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.

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Phosphatase of Regenerating Liver-1 Promotes Cell Migration and Invasion and Regulates Filamentous Actin Dynamics

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.110.167809 ◽

2010 ◽

Vol 334 (2) ◽

pp. 627-633 ◽

Cited By ~ 19

Author(s):

Masanao Nakashima ◽

John S. Lazo

Keyword(s):

Cell Migration ◽

Actin Dynamics ◽

Migration And Invasion ◽

Regenerating Liver ◽

Filamentous Actin ◽

Cell Migration And Invasion ◽

Phosphatase Of Regenerating Liver

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Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

Journal of Biological Chemistry ◽

10.1074/jbc.m113.484535 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20966-20977 ◽

Cited By ~ 51

Author(s):

Haitao Zhang ◽

Pooja Ghai ◽

Huhehasi Wu ◽

Changhui Wang ◽

Jeffrey Field ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Hela Cells ◽

Actin Polymerization ◽

Actin Dynamics ◽

Ras Signaling ◽

Filamentous Actin ◽

Camp Pathway

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

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IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

AJP Cell Physiology ◽

10.1152/ajpcell.00197.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. C256-C263 ◽

Cited By ~ 27

Author(s):

Tatsuya Oka ◽

Masatoshi Hori ◽

Akane Tanaka ◽

Hiroshi Matsuda ◽

Hideaki Karaki ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Cell Activation ◽

De Novo ◽

Immunoglobulin E ◽

Cytochalasin D ◽

Mast Cell Activation ◽

Actin Dynamics ◽

Actin Assembly ◽

Filamentous Actin

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcϵRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca2+ level ([Ca2+]i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca2+]i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca2+]i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca2+]i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca2+]i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca2+ signaling and degranulation.

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Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

The Journal of Cell Biology ◽

10.1083/jcb.201010059 ◽

2011 ◽

Vol 193 (7) ◽

pp. 1289-1303 ◽

Cited By ~ 62

Author(s):

Violaine D. Delorme-Walker ◽

Jeffrey R. Peterson ◽

Jonathan Chernoff ◽

Clare M. Waterman ◽

Gaudenz Danuser ◽

...

Keyword(s):

Cell Migration ◽

Cell Motility ◽

Adhesion Strength ◽

Focal Adhesion ◽

Leading Edge ◽

Actin Dynamics ◽

Filamentous Actin ◽

Downstream Effectors ◽

Myosin Iia ◽

And Migration

Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.

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Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e10-04-0316 ◽

2010 ◽

Vol 21 (20) ◽

pp. 3529-3539 ◽

Cited By ~ 53

Author(s):

Tim Ting Chiu ◽

Nish Patel ◽

Alisa E. Shaw ◽

James R. Bamburg ◽

Amira Klip

Keyword(s):

Actin Polymerization ◽

Molecular Mechanisms ◽

Muscle Cells ◽

Actin Dynamics ◽

Cell Cortex ◽

Glut4 Translocation ◽

Filamentous Actin ◽

Cortical Actin ◽

Rac Gtpase ◽

Skeletal Myoblasts

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

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?2-Adrenoceptor-mediated suppression of human intestinal mast cell functions is caused by disruption of filamentous actin dynamics

European Journal of Immunology ◽

10.1002/eji.200425869 ◽

2005 ◽

Vol 35 (4) ◽

pp. 1124-1132 ◽

Cited By ~ 25

Author(s):

Thomas Gebhardt ◽

Ralf Gerhard ◽

Sammy Bedoui ◽

Veit?J. Erpenbeck ◽

Matthias?W. Hoffmann ◽

...

Keyword(s):

Mast Cell ◽

Actin Dynamics ◽

Filamentous Actin ◽

Cell Functions

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ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2015550117 ◽

2020 ◽

Vol 117 (51) ◽

pp. 32757-32763

Author(s):

Mohammad Foteh Ali ◽

Umma Fatema ◽

Xiongbo Peng ◽

Samuel W. Hacker ◽

Daisuke Maruyama ◽

...

Keyword(s):

Nuclear Migration ◽

Flowering Plants ◽

Actin Dynamics ◽

Double Fertilization ◽

Cellular Mechanisms ◽

Control Female ◽

Regulatory Pathways ◽

Dynamic Movement ◽

Myosin Xi ◽

And Control

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration inArabidopsis thaliana(Arabidopsis) andNicotiana tabacum(tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in theArabidopsiscentral cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

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