TPEN, a Zn2+/Fe2+ chelator with low affinity for Ca2+, inhibits lamin assembly, destabilizes nuclear architecture and may independently protect nuclei from apoptosis in vitro

Abstract

We used Xenopus egg extracts to examine the effects of TPEN, a chelator with strong affinities for Zn2+, Fe2+, and Mn2+, on nuclear assembly in vitro. At concentrations above 1 mM, TPEN blocked the assembly of the nuclear lamina and produced nuclei that were profoundly sensitive to stress-induced balloon-like 'shedding' of nuclear membranes away from chromatin-associated membranes. TPEN-arrested nuclei were also defective for DNA replication, which could be explained as secondary to the lack of a lamina. Imaging of TPEN-arrested nuclei by field emission in-lens scanning electron microscopy (FEISEM) revealed clustered, structurally-perturbed nuclear pore complexes. TPEN-arrested nuclei were defective in the accumulation of fluorescent karyophilic proteins. All detectable effects caused by TPEN were downstream of the effects of BAPTA, a Ca2+/Zn2+ chelator that blocks pore complex assembly at two distinct early stages. Surprisingly, TPEN-arrested nuclei, but not control nuclei, remained active for replication in apoptotic extracts, as assayed by [32P]-dCTP incorporation into high molecular weight DNA, suggesting that TPEN blocks a metal-binding protein(s) required for nuclear destruction during programmed cell death.