Journal of Molecular Biology
Characterization of the Head-to-Tail Overlap Complexes Formed by Human Lamin A, B1 and B2 “Half-minilamin” Dimers
Introduction
Nuclear intermediate filament (IF) proteins, also referred to as lamins, are major structural components of the nuclear envelope of all metazoan cells.1, 2 They interact with a multitude of proteins, both from the inner nuclear membrane and from chromatin.3, 4 Lamins were suggested to be key functional components within the nucleoplasm because mutations in the LMNA gene lead to many different diseases.5, 6, 7, 8 The sequence determination of various vertebrate and invertebrate lamin genes revealed that lamins are highly conserved in evolution, pointing to their basic roles in nuclear functions of metazoan cells.9 Lower vertebrates, such as Hydra attenuata or Caenorhabditis elegans, have only one lamin gene, whereas flies have two genes, classified as A-type and B-type.10 Humans harbor three lamin genes, one coding for the A-type lamin A and its major splice variant lamin C, and two for the B-type lamins, termed lamin B1 and lamin B2. All three of them exhibit an identical domain organization, as depicted for a coiled-coil dimer in Fig. 1a.11, 12, 13 In mammals, amino-terminally truncated “half-lamins” are expressed during germ cell development; i.e. lamin C2 and lamin B3, where the former appears to have essential functions during the meiotic phase of spermiogenesis and the latter during the reshaping of the sperm nucleus in the post-meiotic phase. Lamin C2 replaces lamin A and C, whereas lamin B3 replaces lamin B2. These two unusual “truncated” lamins therefore supplement lamin B1 in a functionally unknown but obviously very distinct manner.14, 15 In any case, these half-lamins provide a proper coil 2, which might associate with lamin B1, provided all lamins can principally associate head-to-tail with one another.
During mitosis, the nuclear lamina disassembles and as a result the A-type lamins are found in the soluble fraction, whereas the B-type lamins stay membrane-bound.16, 17, 18 This disassembly is mediated by phosphorylation through cyclin-dependent kinases and has been described to proceed down to the dimeric level.19, 20 Hence, A- and B-type lamins appear to form homodimers in vivo, but on a higher level of assembly it is not clear whether individual filaments contain both types of lamins within the same filament or if individual lamins segregate into distinct filaments. Moreover, because after completion of mitosis lamin A is incorporated into the reforming lamina some time after B-type lamins, both types of lamins might indeed assemble into distinct filaments.21 Alternatively, lamin A could elongate and enforce existing B-type lamin filament arrays by directly adding on to existing filament ends or by interacting with them laterally.
The lateral exchange of small subunits such as tetramers has been discussed as a principal reaction mediating the dynamics of cytoplasmic IFs.11, 22, 23 Along these lines, a recent high-resolution microscopy study of the distribution of A- and B-type lamins within the nuclear envelope of mammalian somatic cells has indicated that areas of segregation between both types of lamins, as well as areas with an intense overlap, occur side by side.24 Hence, a lateral exchange mechanism might indeed take place, probably regulated by enzymatic mechanisms such as site-specific phosphorylation. Moreover, both types of assembly, homopolymeric as well as heteropolymeric arrays, might provide—depending on the combinations—a meshwork of high functional distinction and thereby modulating regional envelope properties.
The molecular mechanism of IF assembly is not fully understood for the different members of this multigene family. Recently, we proposed that three assembly groups, keratins, vimentin-type proteins and lamins, might be distinguished according to the way these proteins assemble.13, 25 Notably, members of one group do not co-assemble with members of the other groups. In contrast, evolutionarily distantly related proteins of one group can easily co-assemble in vivo and in vitro, as demonstrated for keratins26 and vimentin.27 Keratins are obligate heterodimers of one of two distinct sequence homology classes, whereas the vimentin-like proteins are able to form homopolymers.28 However, when present within the same cells, vimentin assembles into extensive heteropolymer arrays with desmin, GFAP, peripherin, nestin and the neurofilament triplet proteins. For lamins, the above-mentioned in vivo data suggest that A-type and B-type form at least homodimers if not entirely separate filaments. Moreover, it is not known whether the two B-type lamins form homo- or heterodimers.
At the structural level, little is known as to how a lamin filament is organized in comparison to cytoplasmic IFs. Earlier in vitro assembly experiments demonstrated that lamins can form filaments that resemble bona fide cytoplasmic IFs, although they do not form stable 10 nm filaments but instead increase their diameter further by lateral association of filaments over time.1, 29, 30, 31, 32, 33, 34, 35, 36 However, it has been reported that the single B-type lamin of the nematode Caenorhabditis elegans is able to form stable extended and regular 10 nm filaments in vitro.37 Nevertheless, this is not a prominent feature of its assembly and might occur only under certain conditions for a transitory phase.38 Indeed, the strong lateral affinity of lamins for each other is a caveat for the establishment of single elongated filaments.38, 39 It is interesting to note that some mutations in the first 10 heptads of the rod domain could also prevent head-to-tail longitudinal association steps and instead direct assembly to a pathway with pronounced lateral association.40 Last but not least, recent data from cryo-electron tomography of C. elegans lamin filaments revealed that a fraction of the assembled filaments consists of laterally associated protofilaments, which are made from two anti-parallel oriented, head-to-tail associated coiled-coil strands.41
In this study, we investigated one of the prominent in vitro reactions of lamins, i.e. the head-to-tail association of lamin dimers.29, 31 Following up an idea formulated some time ago,1 we generated half-minilamins of all three human lamin types and analyzed their solution and association behavior by analytical ultracentrifugation. We demonstrated that the individual head and tail fragments form stable soluble complexes with one another. Furthermore, we revealed that A- and B-type fragments interact head-to-tail in all possible combinations. These data are of principal importance for understanding how the nuclear envelope is formed in vivo.
Section snippets
The experimental strategy
In the central α-helical coiled-coil-forming domain of lamins, the end segments at either side of the rod are highly conserved. The so-called IF consensus motifs (IFCM1 and IFCM2, Fig. 2) are located within these segments. These amino acid sequence motifs are present in every IF protein in identical or nearly identical form.11, 42 Moreover, the general organization of the central α-helical rod domain is identical for the different lamin types (Fig. 1a). Notably, the rod is divided into two
Discussion
The original idea to use half-minilamins for the structural characterization of the head-to-tail interaction of lamins led us to experiments that revealed a new fundamental result with significant relevance for the description of nuclear lamina formation: the human lamins A, B1 and B2 are able to form hetero-tetrameric complexes in all possible combinations (Fig. 5). This observation has considerable implications for a mechanistic description of the formation of the lamina. Upon mitosis, B-type
Conclusions
Our in vitro data revealed that the IF consensus motifs at either end of the α-helical rod domain, including conserved sequence motifs of the flanking non-α-helical domains, mediate the basic elongation reaction at the level of the coiled-coil dimer between all of the three kinds of lamins, i.e. lamin A, lamin B1 and lamin B2. This interaction may engage the new formation of parallel coiled coils between the carboxy-terminal end segments of one coiled coil with the amino-terminal segments of
Preparation of recombinant lamin fragments
Earlier, we used the prokaryotic expression vector pPEP-T for the cloning and expression of the various lamin fragments.44, 45 However, some of the carboxy-terminal rod fragments were partly cleaved by thrombin after prolonged incubation, so we have used a slightly modified version of pPEP-T denoted pPEP-TEV. In this vector, the codons for the thrombin recognition site were exchanged by the corresponding codons generating the amino acid motif Glu-Asn-Leu-Tyr-Phe-Gln-Gly immediately in front of
Acknowledgements
The authors gratefully acknowledge Ariel Lustig for help with the analytical ultracentrifugation and Tatjana Wedig for excellent technical assistance. U.A. and H.H. were supported by the EURO-Laminopathies research project of the European Commission (contract LSHM-CT-2005-018690). H.H. thanks the German-Israeli Foundation for Scientific Research and Development (GIF Grant No. 915-111.13/2006) for support. S.V.S. acknowledges the support of K.U. Leuven (OT grant /07/071). Moreover, U.A. received
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Contributed equally to this work.