Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Abstract

Abstract. Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK z cells with WGA or antibody PIt and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1 . Although PtK z cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.

Assembly of nuclear pore complexes in Xenopus egg extract

“…This finding agrees with the distinctly punctate immunofluorescent pattern in the cytoplasm of mitotic cells seen with a monoclonal antibody against p68 [5]. Obviously, nuclear pore complexes do not completely disintegrate during mitosis but remain at least partly assembled .…”

“…However, p68 26 MC Dabauvalle, U Scheer does not occur in a monomeric form but rather as a larger and stable macromolecular complex containing, besides p68, a defined set of other proteins [11]. This finding agrees with the distinctly punctate immunofluorescent pattern in the cytoplasm of mitotic cells seen with a monoclonal antibody against p68 [5]. Obviously, nuclear pore complexes do not completely disintegrate during mitosis but remain at least partly assembled .…”

“…In the absence of chromatin, annulate lamellae are being formed (E, pathway II). The newly formed nuclei then take up soluble lamin proteins through the pore complexes (triangles in E; see also [6]) . These imported lamins then become integrated into the already existing "minimal lamina" until the final size of the lamina layer is reached (F) .…”

“…The newly formed pore complexes play an essential part in this remigration of nuclear proteins. When pore-mediated transport is inhibited by microinjection of WGA or a porespecific monoclonal antibody (directed aginst p68; see below), daughter nuclei become enclosed by a nuclear envelope with pore complexes but yet remain arrested in a telophase-like situation, ie are unable to enlarge, to decondense their chromosomes and to reform nucleoli [5,6].The rapid and efficient assembly of functional pore complexes is thus of fundamental importance for postmitotic cells to re-establish interphase nuclear organization and to enter the Gl-phase. Nuclear envelope reconstitution involves the coordinate interactions of nuclear membrane vesicles, depolymerized lamina constituents and disassembled pore complex material, all of which are distributed throughout the cytoplasm of mitotic cell.…”

“…The newly formed pore complexes play an essential part in this remigration of nuclear proteins. When pore-mediated transport is inhibited by microinjection of WGA or a porespecific monoclonal antibody (directed aginst p68; see below), daughter nuclei become enclosed by a nuclear envelope with pore complexes but yet remain arrested in a telophase-like situation, ie are unable to enlarge, to decondense their chromosomes and to reform nucleoli [5,6].…”