Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament.
To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.
Fujiwara
Ikuko
I
Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan.
Suetsugu
Shiro
S
Uemura
Sotaro
S
Takenawa
Tadaomi
T
Ishiwata
Shin'ichi
S
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D003598:Cytoskeletal Proteins; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D013314:Stress, Mechanical; D013997:Time Factors; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D010590:Phalloidine