Xenopus Rtn4a is required for de novo NPC formation in vitro. (A) Inhibition of NPC insertion by anti–(α)-Rtn4a but not anti–(α)-Rtn2 antibodies. Preassembled nuclei were incubated with fresh cytosol in the presence of 10 µM of antibodies for 30 min, and pores were visualized by fluorescently labeled WGA. The NPC number of 50 nuclei is plotted. Error bars represent standard deviation. (B) Schematic illustration of dextran influx assay. When preassembled nuclei (N-0) are incubated with mock-depleted cytosol, intact new pores are inserted (orange circles). In contrast, when N-0 nuclei are incubated with WGA-depleted cytosol, new pores form that cannot exclude dextran (blue circles). If RTNs are required for an early step of NPC assembly, pore/hole formation should be blocked and 70-kD dextran should be excluded. (C) Nuclei were assembled with DiIC16(3)-labeled membranes (red) and incubated with WGA-depleted cytosol in the absence (IgG and α-Rtn2) or presence of inhibitory antibodies (α-Rtn4a) or the calcium chelator BAPTA. After a 10-min incubation, fluorescently labeled 70-kD dextran was added (green), and nuclei were immediately imaged using confocal microscopy (n ≥ 20). (D) In the same reactions as C, surface images were taken from unfixed nuclei that did not exclude (IgG and α-Rtn2) or did exclude (α-Rtn4a) 70-kD dextran. Under all conditions, the NEs were visible as flat membrane patches connected to intact ER tubules. Bars, 10 µm.
