A Highlights from MBoC Selection: Nebulin binding impedes mutant desmin filament assembly

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.     

Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

Assembly defects of desmin disease mutants carrying deletions in the alpha-helical rod domain are rescued by wild type protein

Most mutations of desmin that cause severe autosomal dominant forms of myofibrillar myopathy are point mutations and locate in the central alpha-helical coiled-coil rod domain. Recently, two in-frame deletions of one and three amino acids, respectively, in the alpha-helix have been described and discussed to drastically interfere with the architecture of the desmin dimer and possibly also the formation of tetramers and higher order complexes [Kaminska, A., Strelkov, S.V., Goudeau, B., Olive, M., Dagvadorj, A., Fidzianska, A., Simon-Casteras, M., Shatunov, A., Dalakas, M.C., Ferrer, I., Kwiecinski, H., Vicart, P., Goldfarb, L.G., 2004. Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy. Hum. Genet. 114, 306-313.]. Therefore, it was proposed that they may poison intermediate filament (IF) assembly. We have now recombinantly synthesized both mutant proteins and subjected them to comprehensive in vitro assembly experiments. While exhibiting assembly defects when analyzed on their own, both one-to-one mixtures of the respective mutant protein with wild type desmin facilitated proper filament formation. Transient transfection studies complemented this fundamental finding by demonstrating that wild type desmin is also rescuing these assembly defects in vivo. In summary, our findings strongly question the previous hypothesis that it is assembly incompetence due to molecular rearrangements caused by the mutations, which triggers the development of disease. As an alternative, we propose that these mutations cause subtle age-dependent structural alterations of desmin IFs that eventually lead to disease.     

Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca²⁺]_mit, whereas non-aggregate-prone mutations did not decrease [Ca²⁺]_mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca²⁺]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca²⁺]_mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.     

Severe Myopathy Mutations Modify the Nanomechanics of Desmin Intermediate Filaments

Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of bona fide IFs in vitro and in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.     

Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.

Using immunoelectron microscopy it is demonstrated that desmin subunits missing their complete carboxy-terminal domain are incapable of homopolymeric filament formation in vivo. Furthermore it is shown that, in vimentin-containing cells, desmin integrates into preexisting vimentin filaments resulting in desmin/vimentin heteropolymers. Removal of the amino-terminal or both nonhelical end domains of desmin increases Triton X-100 solubility of the mutant desmin subunits. Expression of desmin mutants containing deletions in the C-terminal part of the rod in vimentin-free cells results in an increase of the Triton X-100 solubility too. In contrast, if expressed in vimentin-containing cells, these mutant subunits remain in the Triton X-100 insoluble fraction. Deletion of the nonhelical carboxy-terminal domain only has no effect on solubility. In vimentin-free cells, stably expressed desmin subunits missing their amino-terminal domains display a slightly higher turnover rate compared to wild-type desmin. Transiently expressed desmin subunits missing 18 or more carboxy-terminal residues of the rod domain are rapidly degraded in vimentin-free cells. In vimentin-containing cells, turnover rates were much less pronounced. Finally, by using site-directed mutagenesis, we were able to map specific residues important for de novo filament assembly within the amino-terminal domain and in the conserved part at the C-terminus of the alpha-helical domain.     

Assembly of carboxy-terminally deleted desmin in vimentin-free cells.

Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely.     

Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network formation with the potential to both activate caspase 3 and decrease cell viability

Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (sHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.     

Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy

Desmin (DES) mutations have been recognized as a cause of desmin-related myopathy (OMIM 601419), or desminopathy, a disease characterized by progressive limb muscle weakness and accumulation of desmin-reactive granular aggregates in the myofibers. We have studied three families with skeletal or cardioskeletal myopathy caused by small in-frame deletions in the desmin gene. The newly identified in-frame deletions E359_S361del and N366del alter the heptad periodicity within a critical 2B coiled-coil segment. Structural analysis reveals that the E359_S361 deletion introduces a second stutter immediately downstream of the naturally occurring stutter, thus doubling the extent of the local coiled-coil unwinding. The N366del mutation converts the wild-type stutter into a different type of discontinuity, a stammer. A stammer, as opposed to a stutter, is expected to cause an extra overwinding of the coiled-coil. These mutations alter the coiled-coil geometry in specific ways leading to fatal damage to desmin filament assembly. Expression studies in two cell lines confirm the inability of desmin molecules with this changed architecture to polymerize into a functional filamentous network. This study provides insights into molecular pathogenetic mechanisms of desmin mutation-associated skeletal and cardioskeletal myopathy.Electronic database information: nucleotide and amino acid sequence data are available in the GenBank database () under accession nos. AY114212 for E359_S361del and AF21879 for N366del mutations     

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.     

Mutations in vimentin disrupt the cytoskeleton in fibroblasts and delay execution of apoptosis

To get new insights into the function of the intermediate filament (IF) protein vimentin in cell physiology, we generated two mutant cDNAs, one with a point mutation in the consensus motif in coil1A (R113C) and one with the complete deletion of coil 2B of the rod domain. In keratins and glia filament protein (GFAP), analogous mutations cause keratinopathies and Alexander disease, respectively. Both mutants prevented filament assembly in vitro and inhibited assembly of wild-type vimentin when present in equal amounts. In stably transfected preadipocytes, these mutants caused the complete disruption of the endogenous vimentin network, demonstrating their dominant-negative behaviour. Cytoplasmic vimentin aggregates colocalised with the chaperones alphaB-crystallin and HSP40. Moreover, vimR113C mutant cells were more resistant against staurosporine-induced apoptosis compared to controls. We hypothesise that mutations in the vimentin gene, like in most classes of IF genes, may contribute to distinct human diseases.     

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.     

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.     

Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.     

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.     

Acute effects of desmin mutations on cytoskeletal and cellular integrity in cardiac myocytes

Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and alpha-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or alpha-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or alpha-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes.     

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

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.