Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain.
We describe a quantitative analysis of Acanthamoeba castellanii myosin II rod domain images collected from atomic force microscope experiments. These images reveal that the rod domain forms a novel filament structure, most likely requiring unusual head-to-tail interactions. Similar filaments are seen also in negatively stained electron microscopy images. Truncated myosins from Acanthamoeba and other model organisms have been visualized before, revealing laterally associated bipolar minifilaments. In contrast, the filament structures that we observe are dominated by axial rather than lateral polymerization. The unusually small features in this structure (1-5 nm) required the development of quantitative and statistical techniques for filament image analysis. These techniques enhance the extraction of features that hitherto have been difficult to ascertain from more qualitative imaging approaches. The heights of the filaments are observed to have a bimodal distribution consistent with the diameters of a single rod domain and a pair of close-packed rod domains. Further quantitative analysis indicates that in-plane association is limited to at most a pair of rod domains. Taken together, this implies that the filaments contain no more than four rod domains laterally associated with one another, somewhat less than that seen in bipolar minifilaments. Analysis of images of the filaments decorated with an anti-FLAG antibody reveals head-to-tail association with mean distances between the antibodies of 75 +/- 15 nm. We consider a set of molecular models to help interpret possible structures of the filaments.
Rigotti
Daniel J
DJ
Department of Biology, Haverford College, 370 Lancaster Ave, Haverford, PA 19041, USA.
Kokona
Bashkim
B
Horne
Theresa
T
Acton
Eric K
EK
Lederman
Carl D
CD
Johnson
Karl A
KA
Manning
Robert S
RS
Kane
Suzanne Amador
SA
Smith
Walter F
WF
Fairman
Robert
R
D048248:Acanthamoeba castellanii; D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000818:Animals; D018625:Microscopy, Atomic Force / Q000379:methods*; D008970:Molecular Weight; D024462:Myosin Type II / Q000737:chemistry* / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D014461:Ultracentrifugation
D024462:Myosin Type II