visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin – dendra2 fusion proteins可视化肌动蛋白聚合的侵入性巨噬细胞和癌细胞结构使用photoconvertibleβ-actinu2014u2014dendra2融合蛋白.pdfVIP

Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible b-Actin – Dendra2 Fusion Proteins 1 1,2 1 1,2 1,2 Athanassios Dovas , Bojana Gligorijevic , Xiaoming Chen , David Entenberg , John Condeelis , Dianne Cox1,3* 1 Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America Abstract Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable b-actin - Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incor