scholarly journals A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

2014 ◽  
Vol 205 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Cortney C. Winkle ◽  
Leslie M. McClain ◽  
Juli G. Valtschanoff ◽  
Charles S. Park ◽  
Christopher Maglione ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cortical Neurons ◽  
Direct Interaction ◽  
Vesicle Fusion ◽  
Dependent Manner ◽  
Axon Branching ◽  
Spatial Regulation ◽  
Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

scholarly journals TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

2017 ◽  
Author(s):  
Melissa Plooster ◽  
Shalini Menon ◽  
Cortney C. Winkle ◽  
Fabio L. Urbina ◽  
Caroline Monkiewicz ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cortical Neurons ◽  
Total Internal Reflection ◽  
Vesicle Fusion ◽  
Src Family Kinases ◽  
Axon Branching ◽  
Deleted In Colorectal Cancer ◽  
Tripartite Motif

AbstractExtracellular netrin-1 and its receptor DCC promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that TRIM9-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased SNARE-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane were increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.Abbreviations used in this paperDCCDeleted in Colorectal CancerTRIMTripartite MotifSFKsrc family kinaseDCCKRnon ubiquitinatable DCC mutantVAMPvesicle associated membrane proteinTRIM9ΔRINGTRIM9 lacking the ubiquitin ligase RING domainTRIM9ΔSPRYTRIM9 variant lacking the DCC-binding SPRY domainTIRFTotal Internal Reflection FluorescencepYphosphotyrosineFAKipharmacological FAK inhibitor 14FRNKFAK related non-kinaseSTX-1Asyntaxin 1AIPimmunoprecipitate

scholarly journals TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

2017 ◽  
Vol 28 (18) ◽  
pp. 2374-2385 ◽  
Author(s):  
Melissa Plooster ◽  
Shalini Menon ◽  
Cortney C. Winkle ◽  
Fabio L. Urbina ◽  
Caroline Monkiewicz ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Vesicle Fusion ◽  
Src Family Kinases ◽  
Axon Branching ◽  
Attachment Protein ◽  
Deleted In Colorectal Cancer ◽  
Protein Receptor ◽  
Tripartite Motif

Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.

scholarly journals Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association

1998 ◽  
Vol 140 (3) ◽  
pp. 647-657 ◽  
Author(s):  
Takeshi Matsui ◽  
Masato Maeda ◽  
Yoshinori Doi ◽  
Shigenobu Yonemura ◽  
Mutsuki Amano ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Catalytic Domain ◽  
Rho Kinase ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Erm Proteins ◽  
Filament Plasma

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ∼30 and ∼100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

scholarly journals Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

2013 ◽  
Vol 203 (4) ◽  
pp. 673-689 ◽  
Author(s):  
Ah-Lai Law ◽  
Anne Vehlow ◽  
Maria Kotini ◽  
Lauren Dodgson ◽  
Daniel Soong ◽  
...  
Keyword(s):  
Cell Migration ◽  
Neural Crest ◽  
Neural Crest Cell ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Border Cell ◽  
Promote Cell Migration ◽  
Wave Complex

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

scholarly journals KBP interacts with SCG10, linking Goldberg–Shprintzen syndrome to microtubule dynamics and neuronal differentiation

2010 ◽  
Vol 19 (18) ◽  
pp. 3642-3651 ◽  
Author(s):  
Maria M. Alves ◽  
Grzegorz Burzynski ◽  
Jean-Marie Delalande ◽  
Jan Osinga ◽  
Annemieke van der Goot ◽  
...  
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Neuronal Differentiation ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Direct Interaction ◽  
Clinical Disorder ◽  
Neurite Development

Abstract Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo . To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

Multinucleated rat alveolar macrophages express functional receptors for calcitonin

1991 ◽  
Vol 261 (6) ◽  
pp. F1026-F1032 ◽  
Author(s):  
A. Vignery ◽  
M. J. Raymond ◽  
H. Y. Qian ◽  
F. Wang ◽  
S. A. Rosenzweig
Keyword(s):  
Plasma Membrane ◽  
Alveolar Macrophages ◽  
Giant Cells ◽  
Mononuclear Phagocytes ◽  
Inflammatory Reactions ◽  
Calcitonin Gene ◽  
Novel Model

The fusion of mononuclear phagocytes occurs spontaneously in vivo and leads to the differentiation of either multinucleated giant cells or osteoclasts in chronic inflammatory sites or in bone, respectively. Although osteoclasts are responsible for resorbing bone, the functional role of giant cells in chronic inflammatory reactions and tumors remains poorly understood. We recently reported that the plasma membrane of multinucleated macrophages is, like that of osteoclasts, enriched in Na-K-adenosinetriphosphatases (ATPases). We also observed that the localization of their Na-K-ATPases is restricted to the nonadherent domain of the plasma membrane of cells both in vivo and in vitro, thus imposing a functional polarity on their organization. By following this observation, we wished to investigate whether these cells also expressed, like osteoclasts, functional receptors for calcitonin (CT). To this end, alveolar macrophages were fused in vitro, and both their structural and functional association with CT was analyzed and compared with those of mononucleated peritoneal and alveolar macrophages. Evidence is presented that multinucleated alveolar macrophages express a high copy number of functional receptors for CT. Our results also indicate that alveolar macrophages, much like peritoneal, express functional receptors for calcitonin gene-related peptide. It is suggested that multinucleated rat alveolar macrophages offer a novel model system to study CT receptors and that calcitonin may control local immune reactions where giant cells differentiate.

scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

scholarly journals Histidine–Tryptophan-–Ketoglutarate Solution as a Neuroprotective Against Ischemia/Reperfusion Injury

2018 ◽  
Author(s):  
Jiun Hsu ◽  
Chih-Hsien Wang ◽  
Shu-Chien Huang ◽  
Yung-Wei Chen ◽  
Shengpin Yu ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Sterile Saline ◽  
Nadph Oxidase 4 ◽  
H2o2 Level

Ischemic neuron loss contributes to brain dysfunction in patients with cardiac arrest (CA). Histidine–tryptophan–ketoglutarate (HTK) solution is a preservative used during organ transplantation. Can HTK also protect neurons from severe hypoxia (SH) following CA? We isolated rat primary cortical neurons and induced SH with or without HTK. Changes in caspase-3, hypoxia-inducible factor 1-alpha (HIF-1α), and NADPH oxidase-4 (NOX4) expression were evaluated at different time points till 72 h. Using a rat asphyxia model, we induced CA-mediated brain damage and then completed resuscitation. HTK or sterile saline was administered into the left carotid artery. Neurological deficit scoring and mortality were evaluated for 3 days. Then the rats were sacrificed for evaluating NOX4 and H2O2 level in blood and brain. In the in vitro study, HTK attenuated SH- and H2O2-mediated cytotoxicity in a volume- and time-dependent manner, associated with persisted HIF-1α expression, reductions in procaspase-3 activation and NOX4 expression. The inhibition of HIF-1α abrogated HTK’s effect on NOX4. In the in vivo study, neurological scores were significantly improved by HTK. H2O2 level, NOX4 activity and NOX4 gene expression were all decreased in the brain specimen of HTK-treated rats. Our results suggest that HTK acts as an effective neuroprotective solution.

scholarly journals An XA21-Associated Kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors

2013 ◽  
Author(s):  
Xuewei Chen ◽  
Shimin Zuo ◽  
Benjamin Schwessinger ◽  
Mawsheng Chern ◽  
Patrick Canlas ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Yeast Two Hybrid System ◽  
Intracellular Domains ◽  
Activity Dependent

The rice XA21 immune receptor kinase and the structurally related XA3 receptor, confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast-two-hybrid system in a kinase activity dependent manner. OsSERK2 undergoes bidirectional trans-phosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. Taken together, these results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function.

scholarly journals Modification of Thrombin-Induced Platelet Aggregation by Estrogen

1977 ◽  
Author(s):  
H. Nagasawa ◽  
M. Steiner ◽  
M. Baldini
Keyword(s):  
Direct Interaction ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
At Iii ◽  
Platelet Concentration ◽  
Considerable Controversy ◽  
Minimal Concentration ◽  
Induced Aggregation

The effect of estrogens on platelet function has remained a subject of considerable controversy. Neither in vivo nor in vitro studies have yet established a basis for a possible mode of action of this hormone on platelets. Our studies were predicated on previous results obtained by one of us suggesting a direct interaction of estrogens with antithrombin III(AT III), Platelets were isolated by conventional method from freshly drawn blood of volunteers, washed twice with Ca2+-free Tyrode buffer and finally suspended in this medium at a concentration of 4.5 × 105 platelets/mm3. Aggregation was induced by addition of 0.01 units of purified bovine thrombin (390 NIH units/mg protein). Aggregation was immediate reaching a maximum within 1.5-2 min.AT III purified from human plasma (2 U/mg protein) inhibited thrombin-induced aggregation in a predictable, concentration-dependent manner. Addition of 0.06 U AT III produced almost complete inhibition. The inhibiting effect of AT III was found to be related to the platelet concentration. Increasing the latter diminished progressively the effect of AT III on thrombin-induced aggregation.Beta-estradiol also inhibited the AT III effect on thrombin-induced aggregation abolishing it at a concentration of 5 × 10-6M. The minimal concentration of β-estradiol which produced a recognizable effect in this system was 5 × 10-9M. These results indicate a direct effect of estrogen on AT III, modifying the protein in such a way that subsequent interaction with thrombin either becomes impossible or does not lead to the inactivation of the enzyme. In addition a possible neutralization of AT III by intact platelets is suggested from our data.These studies were supported by a contract from the AEC.

Sign in / Sign up

Export Citation Format

Share Document