P446L-importin-beta inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules

The P446L mutant Drosophila importin-beta (P446L-imp-beta) has been reported to prohibit--in dominant negative fashion--nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-beta we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-beta. A NE also forms over importin-beta-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-beta as efficiently recruits NE vesicles as normal importin-beta. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-beta appears to be related to icreased--as compared to normal importin-beta--microtubule (MT) binding ability of P446L-imp-beta. While wild-type importin-beta is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-beta cannot be removed from the MTs by RanGTP. P446L-imp-beta, like normal importin-beta, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-beta on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.     

Nup53 is required for nuclear envelope and nuclear pore complex assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.     

GTP hydrolysis by Ran is required for nuclear envelope assembly

Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.     

Spatial and temporal control of nuclear envelope assembly by Ran GTPase

Using evidence derived primarily from studies using Xenopus egg extracts, a model for the role of Ran in multiple stages during NE assembly can be proposed (Figure 2). Ran is concentrated on chromatin prior to NE assembly and recruits RCC1 that generates Ran-GTP locally. Recruitment of RCC1 to chromatin may be a specialized mechanism to initiate NE assembly following fertilization of the egg, whereas in somatic cells, RCC1 may be present on chromatin throughout mitosis. Ran-GTP recruits vesicles to the surface of chromatin, and promotes vesicle fusion to form the double membrane of the NE. Ran-GTP may recruit membrane vesicles to chromatin through binding to integral membrane proteins through importin-beta. A transient complex would be formed between Ran-GTP, importin-beta and the target protein, which would be released locally to promote assembly of a precursor complex. GTP hydrolysis by Ran would release importin-beta, but may also play a role in vesicle fusion. Ran-GTP also promotes NPC assembly by releasing nucleoporins such as Nup107 from inhibitory complexes with importin-beta. In vertebrate cells undergoing mitosis, the majority of Ran molecules are excluded from the chromosomes and dispersed into the cytoplasm. Relocalization of Ran to chromatin at the end of mitosis may co-ordinate the initiation of NE assembly with disassembly of the mitotic spindle. The function of Ran in this transition is likely to be coupled to changes in the activity of cyclin-dependent protein kinases and other activities that control the progression of the cell cycle. Thus, changes in the localization of Ran and its regulators provide temporal and spatial control of NE assembly at the end of mitosis.     

A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro

The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease-sensitive component(s) is resistant to treatments that disrupt protein-protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function.     

Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.     

Role of importin-beta in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

Tyrosine kinase regulation of nuclear envelope assembly

We have described the co-localization of PLCγ and SFK1/7 in vivo and in vitro, and demonstrated their recruitment together to nuclei during membrane binding. We hypothesize that SFK1/7 is activated in response to GTP hydrolysis, phosphorylating and priming PLCγ for activation and thus producing the fusogen DAG during NE assembly. In addition to enhancing our understanding of the temporal and spatial mechanisms of NE formation, this pathway offers a novel link between protein regulation and lipid metabolism that has implications for general membrane fusion events.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly. Project supported by the National Natural Science Foundation of China.     

Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles

Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.     

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

Yeast nuclear pore complex assembly defects determined by nuclear envelope reconstruction

Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understood cellular function. One approach to studying NPC assembly is to identify yeast mutants defective in this process. This requires robust assays for NPC assembly that can be used for phenotypic analysis. We have previously reconstructed yeast nuclei from electron micrographs of serially sectioned cells to precisely determine the number of NPCs (Winey et al., 1997). Here we report the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96 (Zabel et al., 1996) and NUP192 (Kosova et al., 1999). Using conditional alleles of either gene, we have found that the NPC number falls significantly following shift to the restrictive temperature. We conclude that the drop in NPC number results from the failure to assemble new NPCs during cell divisions, leading to the dilution of NPCs that existed when the cells were shifted to the restrictive temperature. We are also able to document a subtle defect in NPC numbers in nup192-15 cells at their permissive temperature. The data presented here quantitatively demonstrate that NPC numbers fall in nic96-1 and nup192-15 strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assembly.     

Lamin B methylation and assembly into the nuclear envelope

Lamin B is reversibly methyl-esterified and phosphorylated during the mammalian cell cycle. In order to study the role of methylation in lamin B function, we isolated mitotic cells in the presence of the microtubule inhibitor, nocodazole. Following removal of nocodazole, methylation of mitotic lamin B was found to precede its assembly into the nuclear envelope as cells exited mitosis. Very little additional methylation took place on assembled lamins. We were able to slow the rate of lamin B methylation with methylthioadenosine (MTA). A delay in lamin B methylation was accompanied by a corresponding delay in assembly of lamin B into the envelope. The delay was approximately 20-30 min beyond the typical 60-70 min usually required. Assembly of lamins A and C, which are not methylated, was also delayed by MTA, although to a lesser degree, suggesting that an interaction between the lamins is necessary for formation of the nuclear envelope. Chromatin decondensation was also slowed in the presence of MTA. Other inhibitors of methylation which had no inhibitory effect on the methyl esterification of lamin B were tested and found to have no effect on envelope assembly or chromatin decondensation. These results were obtained with Chinese hamster ovary cells as well as with the stem cell line, PC 13. Dephosphorylation of lamin B normally follows a time course similar to that of nuclear envelope assembly. In the presence of MTA, however, lamin B assembly was slowed with little effect on dephosphorylation. This resulted in a large population of dephosphorylated, but unassembled, lamin B protein, demonstrating that dephosphorylation is not sufficient for envelope assembly. The lack of effect on the time course of dephosphorylation also suggests that MTA is not acting upstream of the methylation event.     

Nup358 integrates nuclear envelope breakdown with kinetochore assembly

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.