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© The Rockefeller University Press,
0021-9525/1997//559 $5.00
The Journal of Cell Biology, Volume 138, Number 3,
, 1997 559-574
Article |
A Correlative Analysis of Actin Filament Assembly, Structure, and Dynamics
School of Biological Sciences, University of Auckland, Auckland, New Zealand
The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation– condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The "ragged" and "branched" filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg–F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.
Abbreviations used in this paper: 1,4-PBM,N,N'-1,4-phenylenebismaleimide; 2-D, two-dimensional; 3-D, three-dimensional; ADF, annular dark-field; CTEM and STEM, conventional and scanning transmission electron microscopy; DMF, dimethylformamide; HAS, high-affinity divalent cation binding site; LD and UD, lower and upper dimer.
Please address all correspondence to Ueli Aebi, M.E. Müller Institute for Microscopy, Biozentrum, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 0041-61-267-22 61. Fax: 0041-61-267-22 59. E-mail: aebi{at}ubaclu.unibas.ch
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