scholarly journals Retrograde Transport of Transcription Factor NF-κB in Living Neurons

2000 ◽  
Vol 276 (15) ◽  
pp. 11821-11829 ◽  
Author(s):  
Henning Wellmann ◽  
Barbara Kaltschmidt ◽  
Christian Kaltschmidt
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Receptor Activation ◽  
Transcriptional Changes ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Living Neurons

The mechanism by which signals such as those produced by glutamate are transferred to the nucleus may involve direct transport of an activated transcription factor to trigger long-term transcriptional changes. Ionotropic glutamate receptor activation or depolarization activates transcription factor NF-κB and leads to translocation of NF-κB from the cytoplasm to the nucleus. We investigated the dynamics of NF-κB translocation in living neurons by tracing the NF-κB subunit RelA (p65) with jellyfish green fluorescent protein. We found that green fluorescent protein-RelA was located in either the nucleus or cytoplasm and neurites, depending on the coexpression of the cognate inhibitor of NF-κB, IκB-α. Stimulation with glutamate, kainate, or potassium chloride resulted in a redistribution of NF-κB from neurites to the nucleus. This transport depended on an intact nuclear localization signal on RelA. Thus, in addition to its role as a transcription factor, NF-κB may be a signal transducer, transmitting transient glutamatergic signals from distant sites to the nucleus.

scholarly journals Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

Endocrinology ◽  
2006 ◽  
Vol 147 (7) ◽  
pp. 3190-3195 ◽  
Author(s):  
Kate L. J. Ellacott ◽  
Ilia G. Halatchev ◽  
Roger D. Cone
Keyword(s):  
Green Fluorescent Protein ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Fluorescent Protein ◽  
Physiological Role ◽  
Cell Group ◽  
Melanocortin System ◽  
Peptide Precursor ◽  
Green Fluorescent

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.

scholarly journals A Truncated Progesterone Receptor (PR-M) Localizes to the Mitochondrion and Controls Cellular Respiration

2013 ◽  
Vol 27 (5) ◽  
pp. 741-753 ◽  
Author(s):  
Qunsheng Dai ◽  
Anish A. Shah ◽  
Rachana V. Garde ◽  
Bryan A. Yonish ◽  
Li Zhang ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Dna Binding ◽  
Progesterone Receptor ◽  
Western Blot ◽  
Mitochondrial Membrane ◽  
Fluorescent Protein ◽  
Cellular Respiration ◽  
Mitochondrial Localization ◽  
Localization Signal ◽  
Green Fluorescent

Abstract The cDNA for a novel truncated progesterone receptor (PR-M) was previously cloned from human adipose and aortic cDNA libraries. The predicted protein sequence contains 16 unique N-terminal amino acids, encoded by a sequence in the distal third intron of the progesterone receptor PR gene, followed by the same amino acid sequence encoded by exons 4 through 8 of the nuclear PR. Thus, PR-M lacks the N terminus A/B domains and the C domain for DNA binding, whereas containing the hinge and hormone-binding domains. In this report, we have localized PR-M to mitochondria using immunofluorescent localization of a PR-M-green fluorescent protein (GFP) fusion protein and in Western blot analyses of purified human heart mitochondrial protein. Removal of the putative N-terminal mitochondrial localization signal obviated association of PR-M with mitochondria, whereas addition of the mitochondrial localization signal to green fluorescent protein resulted in mitochondrial localization. Immunoelectron microscopy and Western blot analysis after mitochondrial fractionation identified PR-M in the outer mitochondrial membrane. Antibody specificity was shown by mass spectrometry identification of a PR peptide in a mitochondrial membrane protein isolation. Cell models of overexpression and gene silencing of PR-M demonstrated a progestin-induced increase in mitochondrial membrane potential and an increase in oxygen consumption consistent with an increase in cellular respiration. This is the first example of a truncated steroid receptor, lacking a DNA-binding domain that localizes to the mitochondrion and initiates direct non-nuclear progesterone action. We hypothesize that progesterone may directly affect cellular energy production to meet the increased metabolic demands of pregnancy.

Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Kristina Mayer ◽  
Maria E. Iolyeva ◽  
Ulf Meyer-Grahle ◽  
Klaudia Brix
Keyword(s):  
Transcription Factor ◽  
Green Fluorescent Protein ◽  
Cathepsin B ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Egfp Expression ◽  
Reporter Protein ◽  
Specific Expression ◽  
Green Fluorescent ◽  
Proof Of Principle

Abstract We hypothesized that tissue-specific expression of cathepsin B-enhanced green fluorescent protein (CB-EGFP) can be driven by the A33-antigen promoter that contains positive cis-regulatory elements, including caudal-related homeobox (CDX) binding sites. The intestine-specific transcription factor Cdx1 is crucial for A33-antigen promoter activation and could thereby induce expression of CB-EGFP. This concept was tested by construction of the vector pA33-CathB-EGFP encoding CB-EGFP downstream of the A33-antigen promoter. Its Cdx1 dependence, as an indication of its intestine-specific expression, was tested in Cdx1-negative CHO-K1 cells. Cdx1 expression was achieved upon transfection with pCdx1-DsRed-Express and was indicated by red fluorescence of the simultaneously translated reporter protein. Immunolabeling with Cdx1-specific antibodies showed correct targeting of the transcription factor to its point of action in nuclei of transfected cells. Co-transfection experiments with plasmids pA33-CathB-EGFP and pCdx1-DsRed-Express confirmed the hypothesis that Cdx1 indeed activates CB-EGFP expression in a manner dependent on the A33-antigen promoter. Co-localization with compartment-specific markers and subcellular fractionation confirmed CB-EGFP trafficking along the expected route to endolysosomal compartments. Hence, the A33-antigen promoter represents a potent tool for induction of Cdx1-dependent CB-EGFP expression in vitro. Our proof-of-principle studies confirm the suitability of this approach in visualizing protease transport in Cdx1-positive tissues of the gastrointestinal tract.

Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIbβ3: effect of β3GFP on αIIbβ3 ligand binding

2001 ◽  
Vol 357 (2) ◽  
pp. 529-536 ◽  
Author(s):  
Sébastien PLANÇON ◽  
Marie-Christine MOREL-KOPP ◽  
Elisabeth SCHAFFNER-RECKINGER ◽  
Ping CHEN ◽  
Nelly KIEFFER
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cytoplasmic Tail ◽  
Receptor Activation ◽  
Living Cells ◽  
Cell Surface Expression ◽  
Β3 Integrin ◽  
Green Fluorescent

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a β3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either αIIb or β3 allowed normal expression, heterodimerization, processing and surface exposure of αIIbGFPβ3 and αIIbβ3GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced αIIbβ3 capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged αIIbβ3. GFP-tagged αIIbβ3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125FAK tyrosine phosphorylation similar to wild-type αIIbβ3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to β3, but not to αIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of αIIbβ3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged αIIbβ3 during the early stages of cell attachment and spreading, starting with αIIbβ3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of αIIbβ3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either αIIb or β3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the β3 integrin cytoplasmic tail, rather than the αIIb subunit, plays a major role in αIIbβ3 affinity modulation. With the successful direct visualization of functional αIIbβ3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

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.

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