Dynamic Imaging and Fluctuation Spectroscopy on Single Microvilli in Opossum Kidney Cells by the Modulation Tracking Method

Luca Lanzano,Peter Fwu,Hector Giral,Moshe Levi,Enrico Gratton

doi:10.1016/j.bpj.2009.12.4116

Luca Lanzano, Peter Fwu + Show 3 more

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https://doi.org/10.1016/j.bpj.2009.12.4116

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Abstract

Regulation of renal tubular inorganic phosphate (Pi) transport occurs via the proximal tubular apical brush border membrane (BBM) sodium gradient-dependent Pi (NaPi) cotransport proteins. Distinct families of NaPi cotransporters show differential regulation under dietary and hormonal stimuli, but the way this is accomplished, for instance through localization in distinct BBM micro- or nano-domains and/or preferential interaction with different PDZ proteins, is not yet understood.Crucial information could come from the application of single molecule fluctuation correlation spectroscopies on the BBM of living cultured Opossum Kidney (OK) cells expressing NaPi co-transporters with different GFP constructs. The BBM is composed of many microvilli, several micron long structures with a diameter of about 100nm. The microvilli show a relatively fast motion (in the seconds time scale) that makes the use of fluctuation spectroscopy difficult.None of the current nano-resolution optical methods seems capable of measuring the clustering dynamics of proteins on the surface of rapidly moving microvilli. We developed an optical imaging technique called Modulation Tracking (MT) in which we track the center of mass of the microvillus at an arbitrary point along its length while the laser spot rapidly oscillates perpendicularly to the surface and the changes in the modulation are used to measure the distance of the spot from the fluorescent surface with nanometer resolution. High resolution images of the microvilli can be obtained scanning slowly along the microvillus axis. Since the moving microvillus is always at the center of the orbit, fluorescence image correlation techniques can be applied making the MT a truly dynamic nano-imaging technique.Work supported in part by NIH RO1 DK066029-01A2 (ML, EG), NIH-P41 P41-RRO3155 (EG, PF and LL) and P50-GM076516 (EG, PF and LL).

Highlights

3904-Pos Fluorescence Correlation Spectroscopy for Clinical Testing in Von Willebrand Disease Richard Torres, Michael Levene
Our substrate is composed by a GFP linked to a functional nuclear localization sequence (NLS) and transfected into living CHO-K1 cells: the recombinant NLS-GFP protein can bind to molecular carriers mediating cytoplasm-to-nucleus active import as well as shuttle across the Nuclear pore complexes (NPCs) by passive diffusion
We show that obstacles to molecular flow can be detected and that the pair correlation functions (pCF) algorithm can recognize the heterogeneity of NLS-GFP intracompartment diffusion as well as the presence of barriers to its transport between compartments

Summary

Introduction

3904-Pos Fluorescence Correlation Spectroscopy for Clinical Testing in Von Willebrand Disease Richard Torres, Michael Levene. 3903-Pos In Vivo Imaging of Single-Molecule Translocation through Nuclear Pore Complexes by Pair Correlation Functions Francesco Cardarelli, Enrico Gratton. We recently introduced a method based on pair correlation functions (pCF) which measure the time the same molecule takes to migrate from one location to another within the cell (1).

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