scholarly journals Syndecan-2 induces filopodia and dendritic spine formation via the neurofibromin–PKA–Ena/VASP pathway

2007 ◽  
Vol 177 (5) ◽  
pp. 829-841 ◽  
Author(s):  
Yi-Ling Lin ◽  
Ya-Ting Lei ◽  
Chen-Jei Hong ◽  
Yi-Ping Hsueh
Keyword(s):  
Hippocampal Neurons ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Human Embryonic Kidney Cells ◽  
Spine Formation ◽  
Downstream Signaling

Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2–induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because neurofibromin, a syndecan-2–binding partner, activates the cyclic adenosine monophosphate pathway, the role of neurofibromin in syndecan-2–induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.

scholarly journals Coronin-1 Function Is Required for Phagosome Formation

2005 ◽  
Vol 16 (7) ◽  
pp. 3077-3087 ◽  
Author(s):  
Ming Yan ◽  
Richard F. Collins ◽  
Sergio Grinstein ◽  
William S. Trimble
Keyword(s):  
Actin Polymerization ◽  
Coiled Coil ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Raw 264.7 Cells ◽  
Live Cells ◽  
Raw 264.7 ◽  
Dimerization Domain ◽  
Actin Remodeling ◽  
Downstream Signaling

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.

scholarly journals ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages

2003 ◽  
Vol 161 (6) ◽  
pp. 1143-1150 ◽  
Author(s):  
Florence Niedergang ◽  
Emma Colucci-Guyon ◽  
Thierry Dubois ◽  
Graça Raposo ◽  
Philippe Chavrier
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adp Ribosylation ◽  
Transient Activation ◽  
Endosomal Recycling ◽  
Membrane Protrusions ◽  
Specific Receptors ◽  
Intracellular Vesicles ◽  
Adp Ribosylation Factor

Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.

scholarly journals The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

1998 ◽  
Vol 143 (4) ◽  
pp. 1087-1099 ◽  
Author(s):  
Angeliki Malliri ◽  
Marc Symons ◽  
Robert F. Hennigan ◽  
Adam F.L. Hurlstone ◽  
Richard F. Lamb ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cortical Actin ◽  
A431 Cells ◽  
Membrane Ruffling ◽  
In Vitro Invasion ◽  
Cytoskeletal Rearrangements ◽  
Lamellipodia Formation

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

scholarly journals Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

2001 ◽  
Vol 21 (24) ◽  
pp. 8615-8625 ◽  
Author(s):  
Hyewon Phee ◽  
William Rodgers ◽  
K. Mark Coggeshall
Keyword(s):  
B Cells ◽  
B Cell ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Ex Vivo ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Dominant Negative Mutant ◽  
Mutant Form

ABSTRACT Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcγRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcγRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5′-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcγRII−/− but not gamma chain−/− splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were corecruited to the BCR, regardless of the presence or absence of FcγRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcγRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP−/− animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcγRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

scholarly journals Cortactin and dynamin are required for the clathrin-independent endocytosis of γc cytokine receptor

2004 ◽  
Vol 168 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Nathalie Sauvonnet ◽  
Annick Dujeancourt ◽  
Alice Dautry-Varsat
Keyword(s):  
Rna Interference ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Cytokine Receptor ◽  
Actin Dynamics ◽  
Dominant Negative Mutant ◽  
Cellular Functions ◽  
Receptor Endocytosis ◽  
The Common ◽  
Negative Mutant

Endocytosis is critical for many cellular functions. We show that endocytosis of the common γc cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for γc receptor endocytosis. Strikingly, cortactin is needed for both γc and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.

scholarly journals RyR-mediated Ca2+ release elicited by neuronal activity induces nuclear Ca2+ signals, CREB phosphorylation, and Npas4/RyR2 expression

2021 ◽  
Vol 118 (33) ◽  
pp. e2102265118
Author(s):  
Pedro Lobos ◽  
Alex Córdova ◽  
Ignacio Vega-Vásquez ◽  
Omar A. Ramírez ◽  
Tatiana Adasme ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Neuronal Activity ◽  
Hippocampal Neurons ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Creb Phosphorylation ◽  
Memory Processes ◽  
Element Binding Protein ◽  
Neuronal Stimulation ◽  
Sequential Generation

The expression of several hippocampal genes implicated in learning and memory processes requires that Ca2+ signals generated in dendritic spines, dendrites, or the soma in response to neuronal stimulation reach the nucleus. The diffusion of Ca2+ in the cytoplasm is highly restricted, so neurons must use other mechanisms to propagate Ca2+ signals to the nucleus. Here, we present evidence showing that Ca2+ release mediated by the ryanodine receptor (RyR) channel type-2 isoform (RyR2) contributes to the generation of nuclear Ca2+ signals induced by gabazine (GBZ) addition, glutamate uncaging in the dendrites, or high-frequency field stimulation of primary hippocampal neurons. Additionally, GBZ treatment significantly increased cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation—a key event in synaptic plasticity and hippocampal memory—and enhanced the expression of Neuronal Per Arnt Sim domain protein 4 (Npas4) and RyR2, two central regulators of these processes. Suppression of RyR-mediated Ca2+ release with ryanodine significantly reduced the increase in CREB phosphorylation and the enhanced Npas4 and RyR2 expression induced by GBZ. We propose that RyR-mediated Ca2+ release induced by neuronal activity, through its contribution to the sequential generation of nuclear Ca2+ signals, CREB phosphorylation, Npas4, and RyR2 up-regulation, plays a central role in hippocampal synaptic plasticity and memory processes.

scholarly journals Cadherin activity is required for activity-induced spine remodeling

2004 ◽  
Vol 167 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Ko Okamura ◽  
Hidekazu Tanaka ◽  
Yoshiki Yagita ◽  
Yoshinaga Saeki ◽  
Akihiko Taguchi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dendritic Spines ◽  
Hippocampal Neurons ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Cytochalasin D ◽  
Dominant Negative ◽  
Spine Head ◽  
Green Fluorescent ◽  
Activity Dependent

Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

scholarly journals Molecular mechanisms of invadopodium formation

2005 ◽  
Vol 168 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hideki Yamaguchi ◽  
Mike Lorenz ◽  
Stephan Kempiak ◽  
Corina Sarmiento ◽  
Salvatore Coniglio ◽  
...  
Keyword(s):  
Rna Interference ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
De Novo ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Matrix Degradation ◽  
Degradation Activity

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

scholarly journals The T-Cell Receptor Regulates Akt (Protein Kinase B) via a Pathway Involving Rac1 and Phosphatidylinositide 3-Kinase

2000 ◽  
Vol 20 (15) ◽  
pp. 5469-5478 ◽  
Author(s):  
Elisabeth M. Genot ◽  
Cecile Arrieumerlou ◽  
Gregory Ku ◽  
Boudewijn M. T. Burgering ◽  
Arthur Weiss ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase B ◽  
Active Form ◽  
Cell Receptor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Dominant Negative Mutant

ABSTRACT The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation.

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