Distribution of actin filament lengths and their orientation measured by gel electrophoresis in capillaries.
F-actin was electrophoresed in capillary tubes filled with agarose gel. The use of capillary imparted high resistance on the gel allowing the use of high enough concentration of salts to keep F-actin polymerized, and allowed the application of high electric fields without liberating considerable amount of heat. The intensity profile of the electrophoretic band of F-actin showed a peak, which in 1% agarose in the electric field of 17.8 V cm-1 at 0 degree C, migrated at 3.4 cm hr-1. Microscopic observation of actin filaments extracted from different positions along the gel showed that during electrophoresis filaments distributed themselves in such a manner that the longest polymers migrated slowest and the shortest migrated fastest. Using this observation we calculated the weight and number distributions of filament lengths from corresponding experimental intensity profiles. Phalloidin-labelled F-actin oriented in the gel upon application of an electric field. F-actin showed unusual orientational response: it oriented rapidly when the field was applied, but relaxed very slowly when the field was removed. Orientation of F-actin varied within an electrophoretic band, longest polymers showing the best orientation and short oligomers and monomers not orienting at all. The degree of orientation increased with the size of the electric field. When F-actin was labelled with phalloidin before electrophoresis, it was no longer able to migrate in the gel, but the electric field oriented it in the same way as when it was labelled after the electrophoresis. These results show that the electrophoresis of F-actin in agarose fractionates it according to its length, that by using electrophoresis it is possible to rapidly obtain distribution of filament lengths, and that F-actin migrates in agarose by the process of reptation.
Borejdo
J
J
Baylor Research Foundation, Baylor University Medical Center, Dallas, TX 75226.
Burlacu
S
S
D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D016650:Fluorescein-5-isothiocyanate; D008854:Microscopy, Electron; D009210:Myofibrils / Q000378:metabolism / Q000648:ultrastructure*; D010590:Phalloidine
D000199:Actins; D010590:Phalloidine; D004996:Ethidium; D016650:Fluorescein-5-isothiocyanate