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 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 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced 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-aminophenoxylethane-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.
Goldberg
M W
MW
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, UK.
Wiese
C
C
Allen
T D
TD
Wilson
K L
KL
D000818:Animals; D002455:Cell Division; D002462:Cell Membrane; D003593:Cytoplasm; D003599:Cytoskeleton; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009685:Nuclear Envelope / Q000187:drug effects / Q000648:ultrastructure*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D014981:Xenopus
D014909:Wheat Germ Agglutinins; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004533:Egtazic Acid