Pure F-actin networks are distorted and branched by steps in the critical-point drying method.
Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.
Resch
Guenter P
GP
Department of Cell Biology, Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria.
Goldie
Kenneth N
KN
Hoenger
Andreas
A
Small
J Victor
JV
D000199:Actins / Q000302:isolation & purification / Q000648:ultrastructure*; D000818:Animals; D016477:Artifacts; D002478:Cells, Cultured; D020285:Cryoelectron Microscopy / Q000379:methods*; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D005612:Freeze Drying; D016624:Negative Staining; D013552:Swine
D000199:Actins