Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET

Luca Lanzano,Hector Giral,Moshe Levi,Enrico Gratton

doi:10.1016/j.bpj.2012.11.3756

Luca Lanzano, Hector Giral + Show 2 more

Open Access

PDF Available

https://doi.org/10.1016/j.bpj.2012.11.3756

Copy DOI

Export

Save

Cite

Journal: Biophysical Journal	Publication Date: Jan 1, 2013
Citations: 1	License type: cc-by

Affiliation: University of Colorado Denver

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

Fluorescence & Other Luminescence II 3496-Pos Board B651 Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET Luca Lanzano 1 , Hector Giral 2 , Moshe Levi 2 , Enrico Gratton 1 . University California, Irvine, CA, USA, 2 University Colorado, Denver, CO, USA. Phosphate (Pi) homeostasis is crucial for many cellular processes. Pi absorption in the kidney proximal tubule and in the intestine is mainly mediated by the type II family of the sodium-dependent phosphate co-transporters (NaPi) which are located in the apical membrane microvilli. Interaction with scaffolding PDZ domain containing proteins is thought to play a central role in the regulation of the abundance and activity of the NaPi transporters, in response to dietary and hormonal stimuli. We investigated protein-protein interactions between NaPi and PDZ proteins in cellular models of the proximal tubule and the intestine using the FLIM detection of FRET [1]. Also, we have recently introduced the nSPIRO method which generalizes the principles of Single Particle Tracking to the imaging of extended subcellular structures like the apical membrane microvilli [2]. using this method we can image the microvilli with high resolution even if they are moving. Here we combine the tracking of single microvilli with the phasor analysis of FLIM to quantify FRET interaction at the level of the single microvilli. We explore how the measurement of FRET on single microvilli compares to the measurement performed on the entire apical membrane, and discuss the advan- tages and limitations of this technique. Work supported in part by NIH RO1 DK066029-01A2 (ML, EG), 8P41GM103540 (EG and LL) and P50GM076516 (EG and LL). [1] Giral, Lanzano et al, J Biol Chem (2011) 286, 15032-15042; Giral, Cranston, Lanzano et al, J Biol Chem (2012) epub ahead of print [2] Lanzano L et al, J Biophotonics (2011) 4, 415-42; Lanzano L and Gratton E, Microsc Res Tech (2012) 75, 1253-1264

Highlights

3496-Pos Board B651 Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET Luca Lanzano1, Hector Giral2, Moshe Levi2, Enrico Gratton1. 1University California, Irvine, CA, USA, 2University Colorado, Denver, CO, USA
Archaerhodopsin 3 (AR3) and Archaerhodopsin T (ArchT) are light-driven proton pumps that are used as genetically targetable neuronal silencers and fluorescent sensors of transmembrane potential
Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 and ArchT incorporate into the plasma membranes of E. coli and mammalian cells

Summary

Introduction

3496-Pos Board B651 Subcellular Imaging of Protein-Protein Interactions in Live Epithelial Cells using Single Particle Tracking-FLIM-FRET Luca Lanzano1, Hector Giral2, Moshe Levi2, Enrico Gratton1. 1University California, Irvine, CA, USA, 2University Colorado, Denver, CO, USA. 3494-Pos Board B649 Near-Ir Resonance Raman and Uv-Visible Absorption Kinetic Spectroscopy of Optogenetic Archaerhodopsin Neuronal Silencers: Effects of Membrane Potential John I. Saint Clair1, Sergey Mamaev1, Daniel Russano1, Joel M.

Results

Conclusion