scholarly journals Matrix Rigidity-Dependent Regulation of Ca2+ at Plasma Membrane Microdomains by FAK Visualized by Fluorescence Resonance Energy Transfer

2018 ◽  
Vol 6 (4) ◽  
pp. 1801290
Author(s):  
Tae-Jin Kim ◽  
Lei Lei ◽  
Jihye Seong ◽  
Jung-Soo Suh ◽  
Yoon-Kwan Jang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Matrix Rigidity ◽  
Fluorescence Resonance ◽  
Plasma Membrane Microdomains

scholarly journals Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

1998 ◽  
Vol 142 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A.K. Kenworthy ◽  
M. Edidin
Keyword(s):  
Energy Transfer ◽  
Lipid Rafts ◽  
Fluorescence Resonance Energy Transfer ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

scholarly journals Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

2007 ◽  
Vol 282 (49) ◽  
pp. 35440-35448 ◽  
Author(s):  
Gerardo R. Corradi ◽  
Hugo P. Adamo
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Average Distance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescence Resonance ◽  
Green Fluorescent

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

scholarly journals Human Disease-causing Mutations Disrupt an N-C-terminal Interaction and Channel Function of Bestrophin 1

2009 ◽  
Vol 284 (24) ◽  
pp. 16473-16481 ◽  
Author(s):  
Zhiqiang Qu ◽  
Wei Cheng ◽  
Yuanyuan Cui ◽  
Yuanyuan Cui ◽  
Jie Zheng
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Macular Dystrophy ◽  
Fluorescence Resonance ◽  
Transfer Signal

Mutations in the human bestrophin 1 (hBest1) chloride channel cause Best vitelliform macular dystrophy. Although mutations in its transmembrane domains were found to alter biophysical properties of the channel, the mechanism for disease-causing mutations in its N and C termini remains elusive. We hypothesized that these mutations lead to channel dysfunction through disruption of an N-C-terminal interaction. Here, we present data demonstrating that hBest1 N and C termini indeed interact both in vivo and in vitro. In addition, using a spectrum-based fluorescence resonance energy transfer method, we showed that functional hBest1 channels in the plasma membrane were multimers. Disease-causing mutations in the N terminus (R19C, R25C, and K30C) and the C terminus (G299E, D301N, and D312N) caused channel dysfunction and disruption of the N-C interaction. Consistent with the functional and biochemical results, mutants D301N and D312N clearly reduced fluorescence resonance energy transfer signal, indicating that the N-C interaction was indeed perturbed. These results suggest that hBest1 functions as a multimer in the plasma membrane, and disruption of the N-C interaction by mutations leads to hBest1 channel dysfunction.

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