title: A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.
abstract: The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement) during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.
authors.affiliation: UCLA Cardiovascular Research Laboratory, Departments of Medicine (Cardiology) and Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Calmettes
authors.affiliation: UCLA Cardiovascular Research Laboratory, Departments of Medicine (Cardiology) and Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: James N
authors.initials: JN
authors.lastname: Weiss
mesh_terms: D000465:Algorithms; D000704:Analysis of Variance; D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D015687:Cell Hypoxia; D002470:Cell Survival; D003198:Computer Simulation; D003199:Computer Systems; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D006593:Hexokinase / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D006593:Hexokinase