Destabilization of Green Fluorescent Protein by Substitution of Its Amino-Terminal Residue

2001 ◽  
pp. 6-9 ◽  
Author(s):  
Jessica Lesmana ◽  
Peter Friedl
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Terminal Residue ◽  
Amino Terminal ◽  
Green Fluorescent

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

A novel WD40 protein, CHE-2, acts cell-autonomously in the formation of C. elegans sensory cilia

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4839-4848 ◽  
Author(s):  
M. Fujiwara ◽  
T. Ishihara ◽  
I. Katsura
Keyword(s):  
Green Fluorescent Protein ◽  
Sensory Neurons ◽  
Fluorescent Protein ◽  
Interaction Analysis ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Wd40 Domain ◽  
Green Fluorescent ◽  
Almost All

To elucidate the mechanism of sensory cilium formation, we analyzed mutants in the Caenorhabditis elegans che-2 gene. These mutants have extremely short cilia with an abnormal posterior projection, and show defects in behaviors that are mediated by ciliated sensory neurons. The che-2 gene encodes a new member of the WD40 protein family, suggesting that it acts in protein-protein interaction. Analysis of mutation sites showed that both the amino-terminal WD40 repeats and the carboxyl-terminal non-WD40 domain are necessary for the CHE-2 function. CHE-2-tagged green fluorescent protein is localized at the cilia of almost all the ciliated sensory neurons. Expression of che-2 in a subset of sensory neurons of a che-2 mutant by using a heterologous promoter resulted in restoration of the functions and cilium morphology of only the che-2-expressing neurons. Thus, che-2 acts cell-autonomously. This technique can be used in the future for determining the function of each type of che-2-expressing sensory neuron. Using green fluorescent protein, we found that the extension of cilia in wild-type animals took place at the late embryonic stage, whereas the cilia of che-2 mutant animals remained always short during development. Hence, the abnormal posterior projection is due to the inability of cilia to extend, rather than degeneration of cilia once correctly formed. Expression of che-2 in a che-2 mutant under a heat shock promoter showed that the extension of cilia, surprisingly, can occur even at the adult stage, and that such cilia can function apparently normally in behavior.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein

1999 ◽  
Vol 112 (11) ◽  
pp. 1709-1719 ◽  
Author(s):  
C. Ostlund ◽  
J. Ellenberg ◽  
E. Hallberg ◽  
J. Lippincott-Schwartz ◽  
H.J. Worman
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Muscular Dystrophy ◽  
Nuclear Membrane ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Transmembrane Segment ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Green Fluorescent

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

Transport of Connexins and Gap Junction Turnover in Living Cells Visualized Using Green Fluorescent Protein Tagged Connexins.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1020-1021
Author(s):  
D.W. Laird ◽  
K. Jordan ◽  
P. Fistouris ◽  
J.L. Solan ◽  
P.D. Lampe ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Living Cells ◽  
Adjacent Cell ◽  
Gap Junction Channels ◽  
Amino Terminal ◽  
Type Iv ◽  
Green Fluorescent

Connexins oligomerize into hemichannels, traffic to the cell surface and dock with hemichannels from an adjacent cell to form intercellular gap junction channels. Pulsechase studies have revealed that connexins are subject to a short half-life ranging from 1- 5 hrs. To examine the mechanisms involved in connexin trafficking, gap junction assembly and internalization in living cells we fused green fluorescent protein (GFP) to the amino or carboxyl terminus of full length connexins (Cx). When cDNA encoding Cx43-GFP was transfected into communication-competent NRK cells, Cx43-negative MDCK cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into functional gap junctions (Figure 1). Most notably, the fusion of GFP to the amino terminal of Cx43 (GFP-Cx43) or Cx32 (GFP-Cx32) did not inhibit the co-translational insertion of these polytopic type IV connexins into the membrane and as a result gap junction plaques assembled at cellcell interfaces.

Structure and Targeting of RyR1: Implications from Fusion of Green Fluorescent Protein at the Amino-Terminal

2001 ◽  
Vol 388 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Nancy M. Lorenzon ◽  
Manfred Grabner ◽  
Norio Suda ◽  
Kurt G. Beam
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Amino Terminal ◽  
Green Fluorescent

The Green Fluorescent Protein (GFP)

2013 ◽  
Vol 05 (01) ◽  
pp. 101-102
Author(s):  
Phunlerd Piyaraj
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent
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