Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of Ehrlich ascites tumour cells with triton X-100. I. Biochemical analysis.

Abstract

ABSTRACT Ehrlich ascites tumour cells, propagated in suspension culture, were extracted with Triton X-100 under different ionic conditions to study the sensitivity of intermediate-sized filaments formed in vivo to changes in the ionic environment in vitro. The following results were obtained : (1) in solution of low ionic strength and in the absence of di- and polyvalent cations, vimentin was quantitatively solubilized and recovered from the postnuclear supernatant by precipitation with (NH4),SO4 (23% saturation) or spermidine (2 BIM). (2) When the cells were extracted in low ionic strength buffer in the presence of 4 mM-Mg-acetate or 1 · 2 mM-spermidine, vimentin was quantitatively incorporated into Triton X-100-resistant residual cell structures. It could be easily extracted from Mg2+-stabilized cell residues with Mg2+- and polyamine-free buffer of low salt concentration; spermidine-stabilized cell residues were very resistant to dissociation. (3) In a solution of high salt concentration and in the absence of di- and polyvalent cations, little or no vimentin was retained in residual cell structures. Better, though not quantitative, binding was observed in solution at physiological NaCl or KC1 concentrations. (4) A synergistic effect of mono- and di- or polyvalent cations on the association of vimentin with the nuclei was detected when the extraction of cells was performed at concentrations of these ions, which individually were very inefficient in the formation of residual cell structures. (5) Triton X-100-resistant cell residues formed under any ionic conditions were rather resistant to shearing forces. If release of vimentin occurred, it was due to perturbance of the dissociation equilibrium between bound and free vimentin by dilution or washing. (6) Preincubation of cells in buffers of varying ionic composition and in the absence of Triton X-100, followed by titration of the amount of vimentin still incorporable into residual cell structures by extraction of cells with Triton X-100 in solution of 4 mM-Mg2+, showed that the incorporation of vimentin into cell residues was strictly dependent on the intracellular ionic conditions. (7) The intracellular dissociation of vimentin-containing intermediate-sized filaments, induced by preswelling of cells in low ionic strength buffer, could be reversed, to a considerable extent, by restoration of isotonicity. If the incorporation of vimentin into Triton X-100-resistant residual cell structures is taken as a measure of the intactness of the vimentin-type intermediate-sized filaments, the experimental results demonstrate that the stability of the filaments is largely dependent on the maintenance of physiological concentrations of mono-, di- and polyvalent cations.