NUCLEAR-ENVELOPE ASSEMBLY IN XENOPUS EXTRACTS VISUALIZED BY SCANNING EM REVEALS A TRANSPORT-DEPENDENT ENVELOPE SMOOTHING EVENT

We analyzed the pathway of nuclear envelope assembly in Xenopus egg ex tracts using field emission in-lens scanning electron microscopy, The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail, The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fu lly enclosed by membranes, Confirming previous transmission electron m icroscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') ves icles, many of which bound directly to chromatin, and 'smooth' vesicle s, which appeared to associate primarily with other nuclear vesicles o r membrane patches, The presence of ribosomes, an outer nuclear membra ne marker: on many chromatin-binding vesicles suggested that chromatin -attachment proteins integral to the inner membrane were present on ve sicles that also carried markers of the outer membrane and endoplasmic reticulum, Chromatin-associated vesicles also carried pore membrane p roteins, since pore complexes formed when these vesicles were incubate d with cytosol, A change in nuclear envelope morphology termed 'envelo pe smoothing' occurred 5-15 minutes after enclosure, Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-bin ding nucleoporins, and therefore unable to form functional pore comple xes, remained wrinkled, suggesting that 'smoothing' required active nu clear transport, Lamins accumulated with time when nuclei were enclose d and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes, Very low Bevels of la mins were detected on nuclear intermediates whose surfaces were substa ntially covered with patches of pore-complex-containing envelope, sugg esting that pore complexes might be functional before enclosure.