Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses

Zaccolo, Manuela

doi:10.1161/01.res.0000123825.83803.cd

Cited by 91 publications

(70 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This method takes advantage of spatial separation of subcellular events and provides unambiguous temporal correlation of these events. We believe that this methodology complements multicolor imaging (41,42) and is well suited for simultaneously monitoring multiple signaling events and for evaluating the information flow within signaling cascades or crosstalk between different pathways (43). By using this method, we showed that although cAMP production at the plasma membrane immediately produces a pool of cAMP in the nucleus, this pool is not sufficient to generate functional consequences through PKA on the same timescale.…”

Section: Discussionmentioning

confidence: 93%

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

DiPilato

Cheng

Zhang

2004

Proc. Natl. Acad. Sci. U.S.A.

415

394

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 93%

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

DiPilato

Cheng

Zhang

2004

Proc. Natl. Acad. Sci. U.S.A.

415

394

View full text Add to dashboard Cite

show abstract

“…In this method, FRET is typically measured as the ratio of acceptor emission to donor emission upon excitation of the donor, resulting in a value that is proportional to the degree of physical association between the two fluorphores (11). Correction coefficients are used to compensate for spectral bleed through artifacts (12).…”

Section: Resultsmentioning

confidence: 99%

An essential role for cortactin in the modulation of the potassium channel Kv1.2

Williams¹,

Markey²,

Doczi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Ion channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the potassium channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.actin ͉ cytoskeleton ͉ endocytosis ͉ excitability ͉ trafficking S uppression of Kv1.2 mediated by the M1 muscarinic receptor was the first example of tyrosine phosphorylation-dependent modulation of voltage-dependent ion channels. Later studies revealed that channel suppression involves the dynamic interaction of Kv1.2 with the actin-binding protein cortactin. Cortactin binds to the N and C termini of Kv1.2, and binding is diminished upon tyrosine phosphorylation of the channel (1). That evidence, along with mutagenesis studies, led to the hypothesis that cortactin was necessary for Kv1.2 function, and that its dissociation contributed to channel suppression. Subsequently, we found that the mechanism for channel suppression involves the dynamin-dependent endocytosis of Kv1.2 (2). That finding is particularly intriguing because cortactin's role in endocytosis is becoming increasingly apparent (3, 4).Since cortactin's discovery, its functions have been correlated with specific structural domains. Cortactin was first described as an actin cross-linking protein enriched within the cell cortex (5), a property requiring the fourth cortactin repeat region. Later studies identified cortactin as an activator of the Arp2/3 complex, a property dependent on a tryptophan residue (W22) within cortactin's N terminus (5). Tyrosine phosphorylation of cortactin's C-terminal region by Src family kinases affects its ability to regulate actin (6), identifying cortactin as a link between tyrosine kinase sign...

show abstract

“…FRET is based on the distance-dependent energy transfer form neighbouring donor fluorophore and acceptor fluorophore pairs, which are appropriately orientated and have sufficient overlap between their emission and excitation spectra (Zaccolo 2004). FRET causes a decrease in donor fluorescence intensity and lifetime, and an increase in acceptor emission intensity.…”

Section: Green Fluorescent Protein (Gfp) Imagingmentioning

confidence: 99%

Molecular imaging perspectives

Cassidy

Radda

2005

J. R. Soc. Interface.

136

131

View full text Add to dashboard Cite

Molecular imaging is an emerging technology at the life science/physical science interface which is set to revolutionize our understanding and treatment of disease. The tools of molecular imaging are the imaging modalities and their corresponding contrast agents. These facilitate interaction with a biological target at a molecular level in a number of ways. The diverse nature of molecular imaging requires knowledge from both the life and physical sciences for its successful development and implementation. The aim of this review is to introduce the subject of molecular imaging from both life science and physical science perspectives. However, we will restrict our coverage to the prominent in vivo molecular imaging modalities of magnetic resonance imaging, optical imaging and nuclear imaging. The physical basis of these imaging modalities, the use of contrast agents and the imaging parameters of sensitivity, temporal resolution and spatial resolution are described. Then, the specificity of contrast agents for targeting and sensing molecular events, and some applications of molecular imaging in biology and medicine are given. Finally, the diverse nature of molecular imaging and its reliance on interdisciplinary collaboration is discussed.

show abstract

Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses

Abstract: Abstract-In

Cited by 91 publications

References 68 publications

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

An essential role for cortactin in the modulation of the potassium channel Kv1.2

Molecular imaging perspectives

Contact Info

Product

Resources

About