Authors
Breitsprecher, D. Koestler, S. a. Chizhov, I. Nemethova, M. Mueller, J. Goode, B. L. Small, J. V. Rottner, K. Faix, J.
Affiliations
Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany, Institute of Genetics, University of Bonn, Karlrobert-Kreiten-Strasse 13, D-53115 Bonn, Germany, Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria, Department of Biology and Rosenstiel Basic Medical Science Research Centre, Brandeis University, 415 South Street, Waltham, MA 02454, USA, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
Application Area
Cell Biology
TIRFM
Abstract
Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascin cross-linked actin filaments in vitro and in live cells. By multicolour total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin cross-linked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immuno-labelling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.
Extract
… “fluorescence microscope equipped with an LED light source (CoolLed, Andover, UK) and a rear-illuminated cooled CCD Micromax camera (Roper Scientific).”…
Product Associated Features
pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
Diascopic Technique
-
Live Cell Issues
-
Product Type
Journal
Journal of Cell Science
Year of Publication
2011
Country of Publication
Germany