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Direct real-time observation of actin filament branching mediated by Arp2 3 complex

Summary: Direct real-time observation of actin filament
branching mediated by Arp2 3 complex
using total internal reflection
fluorescence microscopy
Kurt J. Amann* and Thomas D. Pollard*
*The Salk Institute for Biological Studies, Structural Biology Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037; and Department of
Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520
Contributed by Thomas D. Pollard, October 18, 2001
Existing methods for studying actin filament dynamics have al-
lowed analysis only of bulk samples or individual filaments after
treatment with the drug phalloidin, which perturbs filament dy-
namics. Total internal reflection fluorescence microscopy with
rhodamine-labeled actin allowed us to observe polymerization in
real time, without phalloidin. Direct measurements of filament
growth confirmed the rate constants measured by electron mi-
croscopy and established that rhodamine actin is a kinetically
inactive tracer for imaging. In the presence of activated Arp2 3
complex, growing actin filaments form branches at random sites
along their sides, rather than preferentially from their barbed ends.
Actin filament polymerization produces forces that push


Source: Amann, Kurt - Molecular and Cellular Pharmacology Training Program & Department of Zoology, University of Wisconsin at Madison


Collections: Biology and Medicine