Chapter 17 Analysis of Nuclear Envelope Assembly Using Extracts of Xenopus Eggs

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69-90 of LBR sequences are required to bind with importin β at aa 45-462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34(cdc2) kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G(1) phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69-73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase.

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

BackgroundThe nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.ResultsUsing transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling.ConclusionThis work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer’s disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.

Beneath the inner nuclear membrane lies the dense meshwork of the nuclear lamina, which provides structural support for the nuclear envelope and serves as an important organizing center for a number of nuclear and cytoplasmic constituents and processes. Herpesviruses have a significant and wide-ranging impact on human health, and their capacity to replicate and cause disease includes events that occur in the host cell nucleus. Herpesviruses begin assembly of progeny virus in the nuclei of infected cells and their capsids must escape the confines of the nucleus by budding through the inner nuclear membrane (INM) to proceed with later stages of virion assembly and egress. Access of viral capsids to the INM thus necessitates disruption of the dense nuclear lamina layer. We review herpesvirus effects on the nuclear lamina and in particular the roles of the herpes simplex virus-encoded nuclear envelope complex and viral kinases on lamin phosphorylation, dissociation, and nucleocapsid envelopment at the INM.

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

Lamins are the major components of the nuclear lamina, a two-dimensional filamentous network at the periphery of the nucleus in higher eukaryotes, directly underlying the inner nuclear membrane. Several integral proteins of the inner nuclear membrane bind to lamins and may link the nuclear membrane to the core lamina network. The lamins and the lamin-binding proteins lamina-associated polypeptide (LAP)2beta and lamin B receptor (LBR) have been described to bind to DNA or to interact with chromatin via histones, BAF-1, and HP1 chromodomain proteins, respectively, and may provide anchorage sites for chromatin fibers at the nuclear periphery. In addition, lamin A structures on intranuclear filaments, or lamin B in replication foci have been described in the nuclear interior, but their specific roles remain unclear. An isoform of the LAP2 protein family, LAP2alpha, has been found to colocalize with A-type lamins in the nucleoplasm and might be involved in intranuclear structure organization. In the course of cell-cycle-dependent dynamics of the nucleus in higher eukaryotes, lamins as well as lamin-binding proteins seem to possess important functions during various steps of post-mitotic nuclear reassembly, including cross-linking of chromatides, nuclear membrane targeting, nuclear lamina assembly, and the formation of a replication-competent nucleus.

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.

Maintenance of nuclear architecture is crucial for gene regulation, cell proliferation and tissue development. However, during every open mitosis and meiosis, chromosomes are exposed to cytoskeletal forces until they are fully reassembled into mature nuclei. Here we discuss our recent study of nuclear assembly in Xenopus egg extracts, where we showed that the DNA binding protein Developmental pluripotency associated 2 (Dppa2) directly inhibits microtubule polymerization during nuclear formation, and that this is essential for normal nuclear shape and replication. We explore mechanisms by which microtubule dynamics could regulate nuclear formation and morphology, and discuss the importance of both spatial and temporal regulation of microtubules in this process. Moreover, expression of Dppa2 is limited to the early embryo and pluripotent tissues, and we highlight the specific demands of mitosis in these often rapidly dividing cells, in which telophase nuclear assembly must be expedited and may facilitate developmental changes in nuclear architecture.

A cell free system to study reassembly of the nuclear envelope at the end of mitosis

In mammals, the successful development of live offspring by somatic cell nuclear transfer (SCNT) has demonstrated the ability of oocyte or egg cytoplasm to reprogram the differentiated status of somatic DNA. However, the efficiency of development is low, and this has been attributed to incomplete or inappropriate reprogramming of epigenetic status. One such epigenetic marker is methylation of genomic DNA at CpG islands. In SCNT, derived embryo abnormal DNA methylation patterns have been reported by a number of groups; in particular, it has been observed that the methylation pattern of embryonic cells resembles that of the donor cell (Santos et al. 2003 Curr. Biol. 13, 1116-1121). One strategy to improve reprogramming and, hence, development is to erase or reprogram the epigenetic status of the donor cell prior to nuclear transfer. We have previously reported that Xenopus egg and oocyte extracts show a differential effect on transcription. In oocyte extracts Pol I and II transcripts are maintained in the somatic cells; in egg extracts, these are abolished (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). To extend these studies, we have investigated the ability of oocyte and egg extracts to demethylate bovine somatic DNA. Preparation of Xenopus oocyte and egg extracts, culture, permeabilization of donor cells, and incubation conditions were all as previously described (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). Cells were incubated in extracts for 1 and 3 h at 21�C, centrifuged onto glass slides fixed in 4% Para formaldehyde for 15 min, followed by 4 M HCL for 1 h at 39�C, and blocked for 1 h. Cells were stained with mouse monoclonal anti-1MeC (1:50) overnight at 4�C followed by FITC-conjugated goat anti-mouse antibody (1:20) for 1 h at room temperature and mounted in Vectashied containing 10 �g of propidium iodide/mL. Nuclei were scored as positive or negative for 5MeC staining. In control cells, 90% of nuclei stained positively for 5MeC. In both oocyte and egg extracts the number of positive nuclei decreased with time showing demethylation of the somatic DNA 68 and 58% and 38 and 42% positive, respectively, after 1 and 3 h of incubation. Addition of apyrase (2%) to hydrolyze ATP inhibited demethylation in both extracts (90% nuclei positive). High rates of DNA replication were observed in somatic cells in egg extracts in contrast to no replication in oocyte extracts. Aphidicolin (1 �g/20 �L) was added to egg extracts to inhibit DNA replication, and under these conditions, DNA demethylation was abolished, suggesting a passive DNA demethylation mechanism as a result of DNA replication. In conclusion, Xenopus laevis oocyte and egg extracts can demethylate mammalian somatic DNA in an energy-dependent manner. In oocyte extracts, demethylation is independent of DNA replication, suggesting an active mechanism. In egg extracts, DNA replication is required, suggesting a passive mechanism. These studies further demonstrate the differences in reprogramming activities between oocyte and egg cytoplasm and suggest that interspecies extracts may provide a tool for nuclear reprogramming.