title: Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion.
abstract: The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE's success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE's direct measurement and ease of use make it appealing for cell biologists.
authors.affiliation: Physics of Living Systems Group, Massachusetts Institute of Technology, Cambridge, MA 02138, USA; Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, MA 02138, USA.
authors.forename: Rotem
authors.initials: R
authors.lastname: Gura Sadovsky
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel; Alexander Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Shlomi
authors.initials: S
authors.lastname: Brielle
authors.affiliation: Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel. Electronic address: dan@cc.huji.ac.il.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kaganovich
authors.affiliation: Physics of Living Systems Group, Massachusetts Institute of Technology, Cambridge, MA 02138, USA. Electronic address: jengland@mit.edu.
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: England
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism*; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008027:Light; D010777:Photochemistry / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012996:Solutions
chemical_list: D011506:Proteins; D012996:Solutions; D049452:Green Fluorescent Proteins
keywords: Sod1; anomalous diffusion; fluorescence-based techniques; molecular crowding; molecular diffusion; photo-conversion; protein aggregation; protein mobility; protein oligomerization; protein quality control