Lamin A precursor induces barrier-to-autointegration factor nuclear localization

Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation.In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.     

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.     

Nesprin-1alpha contributes to the targeting of mAKAP to the cardiac myocyte nuclear envelope

Muscle A-kinase anchoring protein (mAKAP) is a scaffold protein found principally at the nuclear envelope of striated myocytes. mAKAP maintains a complex consisting of multiple signal transduction molecules including the cAMP-dependent protein kinase A, the ryanodine receptor calcium release channel, phosphodiesterase type 4D3, and protein phosphatase 2A. By an unknown mechanism, a domain containing spectrin repeats is responsible for targeting mAKAP to the nuclear envelope. We now demonstrate that the integral membrane protein nesprin-1alpha serves as a receptor for mAKAP on the nuclear envelope in cardiac myocytes. Nesprin-1alpha is inserted into the nuclear envelope by a conserved, C-terminal, klarsicht-related transmembrane domain and forms homodimers by the binding of an amino-terminal spectrin repeat domain. Through the direct binding of the nesprin-1alpha amino-terminal dimerization domain to the third mAKAP spectrin repeat, nesprin-1alpha targets mAKAP to the nuclear envelope. In turn, overexpression of these spectrin repeat domains in myocytes can displace mAKAP from nesprin-1alpha.     

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.     

Dynein at the nuclear envelope

Most cellular organelles are positioned through active transport by motor proteins. The authors discuss the evidence that dynein has important cell cycle-regulated functions in this context at the nuclear envelope.Most cellular organelles are positioned through active transport by motor proteins. This is especially important during cell division, a time when the organelles and genetic content need to be divided equally between the two daughter cells. Although individual proteins can attain their correct location by diffusion, larger structures are usually positioned through active transport by motor proteins. The main motor that transports cargoes to the minus ends of the microtubules is dynein. In nondividing cells, dynein probably transports or positions the nucleus inside the cells by binding to the nuclear envelope (NE; Burke & Roux, 2009). However, it appears that dynein also has important cell-cycle-regulated functions at the NE, as it is recruited to the NE every cell cycle just before cells enter mitosis (Salina et al, 2002; Splinter et al, 2010). Here, we discuss why dynein might be recruited to the NE for a brief period before mitosis.During late G2 or prophase the centrosomes separate to opposite sides of the nucleus, but remain closely associated with the NE during separation. This close association is probably mediated through NE-bound dynein, which ‘walks'' towards the minus ends of centrosomal microtubules, thereby pulling centrosomes towards the NE (Splinter et al, 2010; Gonczy et al, 1999; Robinson et al, 1999). We speculate that close association of centrosomes to the NE might have several functions. First, if centrosomes are not mechanically coupled to the NE, centrosome movement during separation will occur in random directions and chromosomes will not end up between the two separated centrosomes. In this scenario, individual kinetochores might attach more frequently to microtubules coming from both centrosomes (merotelic attachments), a defect that can result in aneuploidy, a characteristic of cancer. Second, centrosome-nuclear attachment also keeps centrosomes in close proximity to chromosomes, which might facilitate rapid capture of chromosomes by microtubules nucleated by the centrosomes after NE breakdown. This might not be absolutely essential, as chromosome alignment can occur in the absence of centrosomes. However, the spatial proximity of centrosomes and chromosomes at NE breakdown might improve the fidelity of kinetochore capture and chromosome alignment.In addition, dynein has also been suggested to promote centrosome separation in prophase in some systems (Gonczy et al, 1999; Robinson et al, 1999; Vaisberg et al, 1993), although not in others (Tanenbaum et al, 2008). Perhaps dynein, anchored at the NE just before mitosis, could exert force on microtubules emanating from both centrosomes, thereby pulling centrosomes apart. However, this force could also be produced by cortical dynein and specific inhibition of NE-associated or cortical dynein will be required to test which pool is responsible.Dynein has also been implicated in the process of NE breakdown itself, by promoting mechanical shearing of the NE. Two elegant studies showed that microtubules can tear the NE as cells enter mitosis (Salina et al, 2002; Beaudouin et al, 2002). One possibility is that microtubules growing into the NE mechanically disrupt it. Alternatively, NE-associated dynein might ‘walk'' along centrosomal microtubules and thereby pull on the NE, tearing it apart. However, testing the exact role of dynein in NE breakdown is complicated by the fact that centrosomes detach from the NE on inactivation of dynein and centrosomal microtubules stop growing efficiently into the NE. Thus, selective inhibition of dynein function will also be required to test this idea.Specific recruitment of dynein to the NE just before mitosis clearly suggests a role for dynein at the NE in preparing cells for mitosis. A major role of NE-associated dynein is to maintain close association of centrosomes with the NE during centrosome separation, which might be needed for efficient capture and alignment of chromosomes after NE breakdown, but additionally, NE-associated dynein could facilitate breakdown and contribute to centrosome separation in some systems.     

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.     

Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.     

Action of insulin at the nuclear envelope

Summary Insulin binding sites are present on purified nuclear envelopes from liver and other tissues, and EM autoradiographs and other types of studies indicate that insulin can enter intact target cells and interact with several types of intracellular membranes, including the nuclear envelope. More recent studies indicate that insulin has direct effects on both mRNA efflux from isolated nuclei and nuclear envelope NTPase, the enzyme that regulates mRNA efflux. These studies raise the possibility, therefore, that insulin regulates mRNA levels in target cells by directly influencing nuclear membrane functions as NTPase. Since insulin does not dramatically elevate mRNA levels for all proteins, the question arises as to how insulin selectively increases mRNA for specific mRNAs. One possibility is that there is targeting of specific mRNA molecules for specific pore complexes and that insulin may only influence a certain fraction of the nuclear pores. Thus, continued investigation is needed concerning the role of polypeptide hormones such as insulin in nucleocytoplasmic exchange.     

Import and export at the nuclear envelope

Eukaryotic cells transport a myriad of molecules between the nucleus and cytoplasm and have evolved a number of related biochemical pathways to achieve this, many of which have been elucidated in recent years. One central and common component to all the pathways is the NPC. NPC components appear to play vital roles in transport and the NPC is structurally dynamic, but whether its role is as a facilitator, a controller or both is yet to be decided and awaits further analysis on the role of individual components in specific pathways.     

A novel mechanism of nuclear envelope break-down in a fungus: nuclear migration strips off the envelope

In animals, the nuclear envelope disassembles in mitosis, while budding and fission yeast form an intranuclear spindle. Ultrastructural data indicate that basidiomycetes, such as the pathogen Ustilago maydis, undergo an 'open mitosis'. Here we describe the mechanism of nuclear envelope break-down in U. maydis. In interphase, the nucleus resides in the mother cell and the spindle pole body is inactive. Prior to mitosis, it becomes activated and nucleates microtubules that reach into the daughter cell. Dynein appears at microtubule tips and exerts force on the spindle pole body, which leads to the formation of a long nuclear extension that reaches into the bud. Chromosomes migrate through this extension and together with the spindle pole bodies leave the old envelope, which remains in the mother cell until late telophase. Inhibition of nuclear migration or deletion of a Tem1p-like GTPase leads to a 'closed' mitosis, indicating that spindle pole bodies have to reach into the bud where MEN signalling participates in envelope removal. Our data indicate that dynein-mediated premitotic nuclear migration is essential for envelope removal in U. maydis.     

Nuclear envelope localization of human UNC84A does not require nuclear lamins

The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.     

Lamin A Ser404 is a nuclear target of Akt phosphorylation in C2C12 cells

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.     

Lamin A/C, laminopathies and premature ageing

Lamin A/C belongs to type V intermediate filaments and constitutes the nuclear lamina and nuclear matrix, where a variety of nuclear activities occur. Lamin A/C protein is firstly synthesized as a precursor and is further proteolytically processed by the zinc metallo-proteinase Ste24 (Zmpste24). Lamin A/C mutations cause a series of human diseases, collectively called laminopathies, the most severe of which is Hutchinson Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) which arises due to an unsuccessful maturation of prelamin A. Although the exact underlying molecular mechanisms are still poorly understood, genomic instability, defective nuclear mechanics and mechanotransduction, have been hypothesized to be responsible for laminopathy-based premature ageing. Removal of unprocessed prelamin A (progerin) or rescue of defective DNA repair could be potential therapeutic strategies for the treatment of HGPS in future.     

Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)-1 and -2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin alpha6beta4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.     

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signal has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding to this sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear envelope fraction. ATP and elevated temperatures enhanced the crosslinking of both nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.