scholarly journals Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

2000 ◽  
Vol 11 (4) ◽  
pp. 1213-1224 ◽  
Author(s):  
Christoph Kaether ◽  
Paul Skehel ◽  
Carlos G. Dotti
Keyword(s):  
Membrane Proteins ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Microscopy Study ◽  
Video Microscopy ◽  
Axonal Membrane ◽  
Color Video ◽  
Green Fluorescent ◽  
Living Neurons

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

scholarly journals Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes

2008 ◽  
Vol 294 (1) ◽  
pp. H229-H237 ◽  
Author(s):  
Kristen M. S. O'Connell ◽  
Jennifer D. Whitesell ◽  
Michael M. Tamkun
Keyword(s):  
Hippocampal Neurons ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Recombinant Adenovirus ◽  
Yellow Fluorescent Protein ◽  
K Channel ◽  
Delayed Rectifier ◽  
Transverse Tubules ◽  
Green Fluorescent

The delayed-rectifier voltage-gated K+ channel (Kv) 2.1 underlies the cardiac slow K+ current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile (τ = 252 ± 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (τ = 479 ± 178 s), although its mobility is slower than Kv1.4 (τ1 = 18.9 ± 2.3 s; τ2 = 305 ± 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis

The Analyst ◽  
2017 ◽  
Vol 142 (19) ◽  
pp. 3648-3655
Author(s):  
Azeem Danish ◽  
Sang-Yong Lee ◽  
Christa E. Müller
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Fluorescence Detection ◽  
Gel Electrophoresis ◽  
Fluorescent Protein ◽  
Laser Induced Fluorescence ◽  
Capillary Gel Electrophoresis ◽  
Laser Induced Fluorescence Detection ◽  
Green Fluorescent

A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF).

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

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.

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