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.
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.
Shumaker
D K
DK
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Vann
L R
LR
Goldberg
M W
MW
Allen
T D
TD
Wilson
K L
KL
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D002614:Chelating Agents / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005029:Ethylenediamines / Q000378:metabolism / Q000494:pharmacology*; D005296:Ferrous Compounds / Q000378:metabolism / Q000494:pharmacology; D034882:Lamins; D009687:Nuclear Proteins / Q000378:metabolism*; D014981:Xenopus; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D002614:Chelating Agents; D005029:Ethylenediamines; D005296:Ferrous Compounds; D034882:Lamins; D009687:Nuclear Proteins; D004533:Egtazic Acid; D015032:Zinc; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium