Nckβ Adapter Controls Neuritogenesis by Maintaining the Cellular Paxillin Level

ABSTRACT The SH2/SH3 adapter Nck has an evolutionarily conserved role in neurons, linking the cell surface signals to actin cytoskeleton-mediated responses. The mechanism, however, remains poorly understood. We have investigated the role of Nck/Nckα/Nck1 versus Grb4/Nckβ/Nck2 side-by-side in the process of mammalian neuritogenesis. Here we show that permanent genetic silencing of Nckβ, but not Nckα, completely blocked nerve growth factor-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. By screening for changes among the components reportedly present in complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckβ knockdown, but not Nckα knockdown, neurons. Interestingly, Nckβ knockdown did not affect the paxillin level in glial cells and several other cell types of various tissue origins. Genetic silencing of paxillin blocked neuritogenesis, just like Nckβ knockdown. Reintroducing a nondegradable Nckβ into Nckβ short interfering RNA-expressing PC12 cells rescued paxillin from down-regulation and allowed the resumption of neuritogenesis. Forced expression of paxillin in Nckβ knockdown PC12 also rescued its capacity for neuritogenesis. Finally, Nckβ, but not Nckα, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin down-regulation in Nckβ knockdown neurons. Thus, Nckβ maintains paxillin stability during neuritogenesis.

Download Full-text

Astrocytes Exhibit a Protective Role in Neuronal Firing Patterns under Chemically Induced Seizures in Neuron–Astrocyte Co-Cultures

International Journal of Molecular Sciences ◽

10.3390/ijms222312770 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12770

Author(s):

Annika Ahtiainen ◽

Barbara Genocchi ◽

Jarno M. A. Tanskanen ◽

Michael T. Barros ◽

Jari A. K. Hyttinen ◽

...

Keyword(s):

Cortical Neurons ◽

Microelectrode Arrays ◽

Cell Types ◽

Protective Role ◽

Neuronal Firing ◽

Neuronal Homeostasis ◽

Chemically Induced ◽

Rat Cortical Neurons

Astrocytes and neurons respond to each other by releasing transmitters, such as γ-aminobutyric acid (GABA) and glutamate, that modulate the synaptic transmission and electrochemical behavior of both cell types. Astrocytes also maintain neuronal homeostasis by clearing neurotransmitters from the extracellular space. These astrocytic actions are altered in diseases involving malfunction of neurons, e.g., in epilepsy, Alzheimer’s disease, and Parkinson’s disease. Convulsant drugs such as 4-aminopyridine (4-AP) and gabazine are commonly used to study epilepsy in vitro. In this study, we aim to assess the modulatory roles of astrocytes during epileptic-like conditions and in compensating drug-elicited hyperactivity. We plated rat cortical neurons and astrocytes with different ratios on microelectrode arrays, induced seizures with 4-AP and gabazine, and recorded the evoked neuronal activity. Our results indicated that astrocytes effectively counteracted the effect of 4-AP during stimulation. Gabazine, instead, induced neuronal hyperactivity and synchronicity in all cultures. Furthermore, our results showed that the response time to the drugs increased with an increasing number of astrocytes in the co-cultures. To the best of our knowledge, our study is the first that shows the critical modulatory role of astrocytes in 4-AP and gabazine-induced discharges and highlights the importance of considering different proportions of cells in the cultures.

Download Full-text

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

Stem Cells International ◽

10.1155/2019/7281912 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Thu T. Duong ◽

James Lim ◽

Vidyullatha Vasireddy ◽

Tyler Papp ◽

Hung Nguyen ◽

...

Keyword(s):

Cortical Neurons ◽

Transgene Expression ◽

Fluorescent Protein ◽

Ex Vivo ◽

Cell Types ◽

Egfp Expression ◽

Cell Type ◽

Egfp Gene ◽

Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Download Full-text

Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes

Development ◽

10.1242/dev.128.5.711 ◽

2001 ◽

Vol 128 (5) ◽

pp. 711-722 ◽

Cited By ~ 3

Author(s):

T.E. Rusten ◽

R. Cantera ◽

J. Urban ◽

G. Technau ◽

F.C. Kafatos ◽

...

Keyword(s):

Nervous System ◽

Cell Fate ◽

System Development ◽

Cell Types ◽

Nervous System Development ◽

Dual Function ◽

Evolutionarily Conserved ◽

A Cell ◽

And Migration

Genes of the spalt family encode nuclear zinc finger proteins. In Drosophila melanogaster, they are necessary for the establishment of head/trunk identity, correct tracheal migration and patterning of the wing imaginal disc. Spalt proteins display a predominant pattern of expression in the nervous system, not only in Drosophila but also in species of fish, mouse, frog and human, suggesting an evolutionarily conserved role for these proteins in nervous system development. Here we show that Spalt works as a cell fate switch between two EGFR-induced cell types, the oenocytes and the precursors of the pentascolopodial organ in the embryonic peripheral nervous system. We show that removal of spalt increases the number of scolopodia, as a result of extra secondary recruitment of precursor cells at the expense of the oenocytes. In addition, the absence of spalt causes defects in the normal migration of the pentascolopodial organ. The dual function of spalt in the development of this organ, recruitment of precursors and migration, is reminiscent of its role in tracheal formation and of the role of a spalt homologue, sem-4, in the Caenorhabditis elegans nervous system.

Download Full-text

Novel role of the muskelin–RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation

The Journal of Cell Biology ◽

10.1083/jcb.200801133 ◽

2008 ◽

Vol 182 (4) ◽

pp. 727-739 ◽

Cited By ~ 41

Author(s):

Manojkumar Valiyaveettil ◽

Amber A. Bentley ◽

Priya Gursahaney ◽

Rajaa Hussien ◽

Ritu Chakravarti ◽

...

Keyword(s):

Cell Morphology ◽

Subcellular Fractionation ◽

Cytoplasmic Localization ◽

Intracellular Protein ◽

Morphological Alterations ◽

C Terminus ◽

Evolutionarily Conserved ◽

The Status ◽

Interfering Rna

The evolutionarily conserved kelch-repeat protein muskelin was identified as an intracellular mediator of cell spreading. We discovered that its morphological activity is controlled by association with RanBP9/RanBPM, a protein involved in transmembrane signaling and a conserved intracellular protein complex. By subcellular fractionation, endogenous muskelin is present in both the nucleus and the cytosol. Muskelin subcellular localization is coregulated by its C terminus, which provides a cytoplasmic restraint and also controls the interaction of muskelin with RanBP9, and its atypical lissencephaly-1 homology motif, which has a nuclear localization activity which is regulated by the status of the C terminus. Transient or stable short interfering RNA–based knockdown of muskelin resulted in protrusive cell morphologies with enlarged cell perimeters. Morphology was specifically restored by complementary DNAs encoding forms of muskelin with full activity of the C terminus for cytoplasmic localization and RanBP9 binding. Knockdown of RanBP9 resulted in equivalent morphological alterations. These novel findings identify a role for muskelin–RanBP9 complex in pathways that integrate cell morphology regulation and nucleocytoplasmic communication.

Download Full-text

Functional Analysis of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Differentially Expressed by Variants of Human HT-1080 Fibrosarcoma Exhibiting High and Low Levels of Intravasation and Metastasis

Journal of Biological Chemistry ◽

10.1074/jbc.m705993200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35964-35977 ◽

Cited By ~ 25

Author(s):

Juneth J. Partridge ◽

Mark A. Madsen ◽

Veronica C. Ardi ◽

Thales Papagiannakopoulos ◽

Tatyana A. Kupriyanova ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Cancer Cell ◽

Small Interfering Rna ◽

Cultured Cells ◽

Tissue Inhibitors Of Metalloproteinases ◽

Down Regulation ◽

Primary Tumors ◽

Tissue Inhibitors ◽

Interfering Rna

The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.

Download Full-text

A Role of Peroxides in Ca2+Ionophore-Induced Apoptosis in Cultured Rat Cortical Neurons

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1996.1538 ◽

1996 ◽

Vol 227 (2) ◽

pp. 513-518 ◽

Cited By ~ 13

Author(s):

Yutaka Hatanaka ◽

Keiichiro Suzuki ◽

Yoshimi Kawasaki ◽

Yasuhisa Endo ◽

Naoyuki Taniguchi ◽

...

Keyword(s):

Cortical Neurons ◽

Rat Cortical Neurons ◽

Induced Apoptosis

Download Full-text

Interaction Between IGF Binding Protein-3 and TGFβ in the Regulation of Adipocyte Differentiation

Endocrinology ◽

10.1210/en.2011-1444 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4799-4807 ◽

Cited By ~ 12

Author(s):

Hasanthi C. de Silva ◽

Sue M. Firth ◽

Stephen M. Twigg ◽

Robert C. Baxter

Keyword(s):

Binding Protein ◽

Adipogenic Differentiation ◽

Cell Types ◽

Inhibitory Effect ◽

Down Regulation ◽

Smad2 Phosphorylation ◽

Igf Binding ◽

Igf Binding Protein ◽

Interfering Rna ◽

Igfbp 3

Abstract The development of white adipose tissue involves both the hypertrophy of existing adipocytes and the proliferation and differentiation of preadipocytes. Adipogenic differentiation is inhibited by TGFβ signaling through Smad2/3, and IGF binding protein-3 (IGFBP-3) is also known to activate Smad2/3 signaling in some cell types. We previously reported that exogenous or overexpressed IGFBP-3 inhibits adipogenesis in 3T3-L1 cells, but the role of endogenous IGFBP-3 in this process, and its possible interaction with TGFβ, is not known. During 10-d adipogenic differentiation initiated by insulin, dexamethasone, and 3-isobutyl-1-methylxanthine, 3T3-L1 cells expressed increasing levels of IGFBP-3 and TGFβ1, secreting over 1000 pg/ml of both proteins. Exogenous recombinant human IGFBP-3 paralleled TGFβ1 in stimulating Smad2 phosphorylation in 3T3-L1 preadipocytes, but no additive effect was observed for the two agents. In contrast, knockdown of endogenous IGFBP-3 by small interfering RNA (siRNA) significantly impaired Smad2 activation by 0.25 ng/ml TGFβ1. Transient expression of human IGFBP-3 significantly inhibited the induction of adipogenic markers adiponectin and resistin, and the appearance of lipid droplets, but down-regulation of endogenous IGFBP-3 by siRNA had little effect on the expression of either marker during the 10-d differentiation, compared with nonsilencing control siRNA. However, down-regulation of endogenous IGFBP-3 using two different siRNA significantly reversed the inhibitory effect of TGFβ1 on both adiponectin and resistin induction. We conclude that IGFBP-3 activates inhibitory Smad signaling in 3T3-L1 cells and that endogenous IGFBP-3 modulates their adipogenic differentiation by regulating cell sensitivity towards the inhibitory effect of TGFβ.

Download Full-text

Tribbles in normal and malignant haematopoiesis

Biochemical Society Transactions ◽

10.1042/bst20150117 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1112-1115 ◽

Cited By ~ 13

Author(s):

Sarah J. Stein ◽

Ethan A. Mack ◽

Kelly S. Rome ◽

Warren S. Pear

Keyword(s):

E3 Ligase ◽

Cell Types ◽

Protein Family ◽

Conserved Motifs ◽

Tissue Specific ◽

Cellular Events ◽

Haematopoietic Cells ◽

Evolutionarily Conserved ◽

Multiple Cell

The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types.

Download Full-text

Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0713 ◽

2008 ◽

Vol 19 (2) ◽

pp. 711-721 ◽

Cited By ~ 128

Author(s):

Andras Balla ◽

Yeun Ju Kim ◽

Peter Varnai ◽

Zsofia Szentpetery ◽

Zachary Knight ◽

...

Keyword(s):

Plasma Membrane ◽

Small Interfering Rna ◽

Type Ii ◽

Measurable Effect ◽

Down Regulation ◽

Type Iii ◽

Hormone Sensitive ◽

Phosphatidylinositol 4 ◽

Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

Download Full-text