Journal of Cell Science, Vol 62, Issue 1 419-438, Copyright © 1983 by Company of Biologists

JOURNAL ARTICLES

Selective extraction with Sarkosyl and repolymerization in vitro of cytoskeleton proteins from Giardia

R Crossley and DV Holberton

Sarkosyl has been used to dissociate structures in cytoskeletons isolated from Giardia lamblia. Results from sodium dodecyl sulphate/polyacrylamide gel electrophoresis and from electron microscopy of insoluble residues show that the solubilization of components is partly selective. At low concentrations of detergent (less than 0.3%), microribbons and microtubules of the disc cytoskeleton disappear, but doublet microtubules from axonemes resist extraction. Consequently, the microribbon protein giardin is extracted into solution more completely than tubulin. Soluble proteins in 0.1% Sarkosyl have been fractionated by gel filtration chromatography in Bio-Gel P300. Giardin elutes in two positions: as a low molecular weight subunit, and in early fractions corresponding to a larger particle size in which subunits might be forming oligomers. Supernatants prepared in 0.5% Sarkosyl were diluted with 0.1 M-KCl or 0.1 mM-MgCl2 to bring about reaggregation of the cytoskeleton proteins. Reassembled structures seen in negatively stained preparations were polymorphic. Some tubulin ribbons of 5 nM protofilaments were identifiable' also there were large fibres and some flat sheets of very thin filaments. Electron micrographs of sheets have been analysed by optical diffraction. The transforms show that the lateral separation of the fine filaments is about 2.5 nm. Axial periodicities from features spaced along filaments were weak. A 3.75 nm layer-line has been detected, corresponding to a similar periodicity found earlier in microribbons.