scholarly journals A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes

2008 ◽  
Vol 181 (6) ◽  
pp. 1027-1039 ◽  
Author(s):  
Junghoon Ha ◽  
Kevin W.-H. Lo ◽  
Kenneth R. Myers ◽  
Tiffany M. Carr ◽  
Michael K. Humsi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Receptor Tyrosine Kinase ◽  
Pc12 Cell ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Neurotrophin Receptor ◽  
Immunoaffinity Purification ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

NRSF Causes cAMP-Sensitive Suppression of Sodium Current in Cultured Hippocampal Neurons

2002 ◽  
Vol 88 (1) ◽  
pp. 409-421 ◽  
Author(s):  
H. Nadeau ◽  
H. A. Lester
Keyword(s):  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Sodium Current ◽  
Striking Similarity ◽  
Secondary Effects ◽  
Functional Effect ◽  
Green Fluorescent ◽  
Altered Sensitivity ◽  
Cultured Hippocampal Neurons

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na+channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1–2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na+channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K+currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic “silencer” and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

scholarly journals Roles of NUDE and NUDF Proteins of Aspergillus nidulans: Insights from Intracellular Localization and Overexpression Effects

2003 ◽  
Vol 14 (3) ◽  
pp. 871-888 ◽  
Author(s):  
Vladimir P. Efimov
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Dynein Heavy Chain ◽  
Terminal Domain ◽  
Green Fluorescent

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes (“comets”) that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudEgene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditionalnudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF andnudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between thenudF and apsA genes suggests a role for NUDF at the cell cortex.

scholarly journals Introduction of Green Fluorescent Protein (GFP) into Hippocampal Neurons through Viral Infection

2010 ◽  
Vol 2010 (4) ◽  
pp. pdb.prot5406-pdb.prot5406 ◽  
Author(s):  
R. Malinow ◽  
Y. Hayashi ◽  
M. Maletic-Savatic ◽  
S. H. Zaman ◽  
J.-C. Poncer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Viral Infection ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Propofol Neurotoxicity Is Mediated by p75 Neurotrophin Receptor Activation

2012 ◽  
Vol 116 (2) ◽  
pp. 352-361 ◽  
Author(s):  
Matthew L. Pearn ◽  
Yue Hu ◽  
Ingrid R. Niesman ◽  
Hemal H. Patel ◽  
John C. Drummond ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Receptor Activation ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Wild Type ◽  
P75 Ntr ◽  
Green Fluorescent ◽  
Developing Neurons

Background Propofol exposure to neurons during synaptogenesis results in apoptosis, leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75(NTR)) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75(NTR). Methods Days in vitro 5-7 neurons were exposed to propofol (3 μM) for 6 h and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75(NTR-/-) mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 μM, 15 min), a specific p75(NTR) inhibitor. P75(NTR-/-) neurons were transfected for 72 h with a lentiviral vector containing the synapsin-driven p75(NTR) gene (Syn-p75(NTR)) or control vector (Syn-green fluorescent protein) before propofol. To confirm our in vitro findings, wild-type mice and p75(NTR-/-) mice (PND5) were pretreated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. Results Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75(NTR-/-) neurons. In p75(NTR-/-) neurons transfected with Syn-p75(NTR), propofol significantly increased Cl-Csp3 in comparison with Syn-green fluorescent protein-transfected p75(NTR-/-) neurons. Wild-type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75(NTR-/-) mice. Conclusion These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75(NTR) and the downstream effector RhoA kinase.

scholarly journals Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal Neurons

1999 ◽  
Vol 10 (9) ◽  
pp. 2945-2953 ◽  
Author(s):  
Martin Köhrmann ◽  
Ming Luo ◽  
Christoph Kaether ◽  
Luc DesGroseillers ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Hippocampal Neurons ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Maximum Velocity ◽  
Time Lapse ◽  
Mrna Transport ◽  
Transport Pathway ◽  
Green Fluorescent ◽  
Dependent Transport

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

scholarly journals A Conserved Mechanism of Synaptogyrin Localization

2001 ◽  
Vol 12 (8) ◽  
pp. 2275-2289 ◽  
Author(s):  
Hongjuan Zhao ◽  
Michael L. Nonet
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
C Elegans ◽  
C Terminus ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

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