DIMPLES, PORES, STAR-RINGS, AND THIN RINGS ON GROWING NUCLEAR ENVELOPES - EVIDENCE FOR STRUCTURAL INTERMEDIATES IN NUCLEAR-PORE COMPLEX ASSEMBLY

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear egg extracts, Scattered among comple xes rare 'dimples' (outer membrane depressions, nm diameter), more abu ndant holes (pores) with a variety of edge geometries (35-45 nm diamet er; 3.3% of structures), pores containing one to eight triangular 'sta r-ring' subunits (2.1% of total), and more complicated structures, Nei ther mature complexes, nor these novel structures, formed when wheat g erm agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 mu g/ml) directly to the assembly reaction; low concentrations (10 mu g/ml) had no effect, However at intermediate concentrations (50-100 mu g/ml), wheat germ agglutinin caused a drama tic, sugar-reversible accumulation of 'empty' pores, and other structu res; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-bindi ng nucleoporin, Another inhibitor, dibromo-BAPTA ,2-bis[o-aminophenoxy lethane-N,N,N',N'-tetraacetic acid), had different effects depending o n its time of addition to the assembly reaction, When 1 mM dibromo-BAP TA was added at time zero, no pore-related structures formed, However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after in itiating assembly, star-rings and other structures accumulated, sugges ting that dibromo-BAPTA can inhibit multiple stages in pore complex as sembly, We propose that assembly begins with the formation and stabili zation of a hole (pore) through the nuclear envelope, and that dimples , pores, star-rings, and thin rings are structural intermediates in nu clear pore complex assembly.