Figure 7. Interaction of vimentin with zinc.

(a) Incubation with ZnCl₂ selectively protects vimentin from modification by Iac-B. Polymerized vimentin was incubated with 10 mM EDTA or 500 μM ZnCl₂, CaCl₂ or MgCl₂ before addition of 10 μM Iac-B. Incorporation of Iac-B and vimentin amounts were assessed as in Fig. 1. (b) Effect of zinc and other divalent cations on vimentin polymerization. Vimentin was incubated with the indicated concentrations of ZnCl₂ or with 500 μM MgCl₂, CaCl₂, CuCl₂, ZnCl₂ or NaCl for 1 h at r.t., after which, soluble (S) and polymerized (P) vimentin were assessed as in Fig. 1. The effect of imidazole on vimentin polymerization induced by 500 μM ZnCl₂ or 150 mM NaCl was evaluated as above. Reversion of ZnCl₂-induced vimentin polymerization by TPEN was performed as described in Methods section; n≥3 for all polymerization assays. (c) Analysis of vimentin structures by electron microscopy (EM). Vimentin was incubated with 150 mM NaCl or 500 μM ZnCl₂ for 1 h or with 2.5 mM MgCl₂ o/n, before processing and visualization by EM at 50,000-fold magnification (n≥7). Scale bar, 100 nm. (d) Zinc induces vimentin assembly into optically detectable structures. Vimentin was incubated with 2.5 mM ZnCl₂ at r.t. for 15 min and the structures formed visualized by optical microscopy (n=19). (e) Structures formed upon ZnCl₂ treatment of FITC-labelled vimentin observed by fluorescence confocal microscopy. (f) Labelling of ZnCl₂-induced vimentin filaments with Zinquin (upper panels) and loss of polymerization and Zinquin fluorescence upon TPEN treatment (lower panels). Scale bars, 10 μm, n≥3. (g) Competition assays between vimentin and PAR for zinc binding. Left panel, mixtures containing 100 μM PAR and 1 μM soluble or NaCl-polymerized vimentin were incubated with increasing concentrations of ZnCl₂ and the formation of the ZnPAR₂ complex was monitored by the absorbance at 492 nm. Right panel, effect of magnesium on the ability of vimentin to sequester zinc. Assay mixtures containing PAR or PAR plus vimentin were preincubated with MgCl₂, after which ZnCl₂ was added and the ZnPAR₂ complex was determined at the indicated time points (representative from four assays).