mechanotransduction.org

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare ‘dimples’ (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular ‘star-ring’ subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 mg/ml) directly to the assembly reaction; low concentrations (10 mg/ml) had no effect. However at intermediate concentrations (50-100 mg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of ‘empty’ pores, and other structures; this effect correlated with the lectininduced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5¢-dibromo- 1,2-bis[o-aminophenoxy]ethane-N,N,N¢,N¢-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.