The CaaX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope

While the nuclear lamin proteins (A, B, and C) assemble specifically at the surface of the nuclear membrane, their sequences do not reveal stretches of hydrophobic amino acids that might explain their association with the nuclear membranes. However, the A and B lamin proteins possess Ras-like C-terminal CaaX sequence motifs, which in Ras proteins are sites of hydrophobic modifications required for membrane association and function. From the analysis of single and double lamin A mutants affecting the CaaX motif, the nuclear localization signal, and higher-order assembly properties, we propose that the CaaX motif functions as a nonspecific, low affinity membrane probe for proteins ultimately segregated to specific cellular membrane systems. Committed association with specific membranes requires additional interactions with membrane-resident factors.     

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.     

A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans

Mutations in the human LMNA gene underlie many laminopathic diseases, including Emery-Dreifuss muscular dystrophy (EDMD); however, a mechanistic link between the effect of mutations on lamin filament assembly and disease phenotypes has not been established. We studied the ΔK46 Caenorhabditis elegans lamin mutant, corresponding to EDMD-linked ΔK32 in human lamins A and C. Cryo-electron tomography of lamin ΔK46 filaments in vitro revealed alterations in the lateral assembly of dimeric head-to-tail polymers, which causes abnormal organization of tetrameric protofilaments. Green fluorescent protein (GFP):ΔK46 lamin expressed in C. elegans was found in nuclear aggregates in postembryonic stages along with LEM-2. GFP:ΔK46 also caused mislocalization of emerin away from the nuclear periphery, consistent with a decreased ability of purified emerin to associate with lamin ΔK46 filaments in vitro. GFP:ΔK46 animals had motility defects and muscle structure abnormalities. These results show that changes in lamin filament structure can translate into disease-like phenotypes via altering the localization of nuclear lamina proteins, and suggest a model for how the ΔK32 lamin mutation may cause EDMD in humans.     

Sorting Nexin 6 Enhances Lamin A Synthesis and Incorporation into the Nuclear Envelope

Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A in vitro and in vivo and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.     

A Highlights from MBoC Selection: Concentration-dependent lamin assembly and its roles in the localization of other nuclear proteins

The nuclear lamina (NL) consists of lamin polymers and proteins that bind to the polymers. Disruption of NL proteins such as lamin and emerin leads to developmental defects and human diseases. However, the expression of multiple lamins, including lamin-A/C, lamin-B1, and lamin-B2, in mammals has made it difficult to study the assembly and function of the NL. Consequently, it has been unclear whether different lamins depend on one another for proper NL assembly and which NL functions are shared by all lamins or are specific to one lamin. Using mouse cells deleted of all or different combinations of lamins, we demonstrate that the assembly of each lamin into the NL depends primarily on the lamin concentration present in the nucleus. When expressed at sufficiently high levels, each lamin alone can assemble into an evenly organized NL, which is in turn sufficient to ensure the even distribution of the nuclear pore complexes. By contrast, only lamin-A can ensure the localization of emerin within the NL. Thus, when investigating the role of the NL in development and disease, it is critical to determine the protein levels of relevant lamins and the intricate shared or specific lamin functions in the tissue of interest.     

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.     

Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.     

Structural and physiological phenotypes of disease-linked lamin mutations in C. elegans

The nuclear lamina is a major structural element of the nucleus and is predominately composed of the intermediate filament lamin proteins. Missense mutations in the human lamins A/C cause a family of laminopathic diseases, with no known mechanistic link between the position of the mutation and the resulting disease phenotypes. The Caenorhabditis elegans lamin (Ce-lamin) is structurally and functionally homologous to human lamins, and recent advances have allowed detailed structural analysis of Ce-lamin filaments both in vitro and in vivo. Here, we studied the effect of laminopathic mutations on Ce-lamin filament assembly in vitro and the corresponding physiological phenotypes in animals. We focused on three disease-linked mutations, Q159K, T164P, and L535P, which have previously been shown to affect lamin structure and nuclear localization. Mutations prevented the proper assembly of Ce-lamin into filament and/or paracrystalline arrays. Disease-like phenotypes were observed in strains expressing low levels of these mutant lamins, including decreased fertility and motility coincident with muscle lesions. In addition, the Q159K- and T164P-expressing strains showed a reduced lifespan. Thus, different disease-linked mutations in Ce-lamin exhibit major effects in vivo and in vitro. Using C. elegans as a model system, a comprehensive analysis of the effects of specific lamin mutations from the level of in vitro filament assembly to the physiology of the organism will help uncover the mechanistic differences between these different lamin mutations.     

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.     

Investigation of nuclear architecture with a domain-presenting expression system

We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear vimentin bodies and, vice versa, the LBR chimera attracts lamin B in transfected cells, thereby demonstrating their bona fide interaction in vivo.     

Subcellular localization of SREBP1 depends on its interaction with the C-terminal region of wild-type and disease related A-type lamins

Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (?607–656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.     

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis

The nuclear envelope is a dynamic structure that completely disassembles in response to MPF/cdc2 activity in mitosis. A key feature of this process is the hyperphosphorylation of the major structural proteins of the envelope, the nuclear lamins A, B, and C. Two highly conserved serine residues of the lamin protein (Ser-22 and Ser-392 of lamins A and C) are symmetrically positioned 5 amino acids from the ends of the large alpha-helical domain and are shown in the accompanying paper by Ward and Kirschner to be among four sites phosphorylated during nuclear envelope breakdown. Mutations in Ser-22 and Ser-392 that prevent phosphorylation at these sites block the disassembly of the nuclear lamina during mitosis. We propose a model for the regulation of lamin assembly in which phosphorylation just outside the ends of the alpha-helical domain controls the assembly dynamics of the lamin coiled-coil dimers.     

Solubility properties and specific assembly pathways of the B-type lamin from Caenorhabditis elegans

Lamins are nucleus-specific intermediate filament (IF) proteins that together with a complex set of membrane proteins form a filamentous meshwork tightly adhering to the inner nuclear membrane and being associated with the nuclear pore complexes. This so-called nuclear lamina provides mechanical stability and, in addition, has been implicated in the spatial organization of the heterochromatin. While increasing knowledge on the biological function of lamins has been obtained in recent years, the assembly mechanism of lamin filaments at the molecular level has remained largely elusive. Therefore, we have now more systematically investigated lamin assembly in vitro. Using Caenorhabditis elegans lamin, which has been reported to assemble into 10-nm filaments under low ionic strength conditions, we investigated the assembly kinetics of this protein into filaments in more detail using both His-tagged and un-tagged recombinant proteins. In particular, we have characterized distinct intermediates in the filament assembly process by analytical ultracentrifugation, electron and atomic force microscopy. In contrast to the general view that lamins assemble only slowly into filaments, we show that in vitro association reactions are extremely fast, and depending on the ionic conditions employed, significant filamentous assemblies form within seconds.     

Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics.

The nuclear lamin proteins assemble at the nuclear membrane into a highly dynamic network whose integrity is exquisitely controlled by the major cell cycle regulators. Although the actual cellular functions of the lamins remain elusive, the processes of targeted lamin assembly and phosphorylation-induced disassembly have come to light recently. These processes require functionally interacting lamin domains to execute targeting to the nucleus and the nuclear membranes, lamina assembly, and the disassembly response to cell-cycle-dependent phosphorylation.