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The molecular architecture of lamins in somatic cells

Abstract

The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin1,2,3,4. Besides providing structural stability to the nucleus5,6, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication7,8,9,10. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

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Figure 1: Architecture of the filamentous meshwork of the lamina of the mammalian cell nucleus.
Figure 2: Characterization of the lamin meshwork.
Figure 3: Structural analysis of lamin filaments.
Figure 4: The nuclear lamina and cytoskeletal constituents of mammalian somatic cells.

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Acknowledgements

We thank R. Irobalieva for reading the manuscript, Y. Zheng for providing cell lines and L. Gerace for sharing lamin B1 antibody. This work was funded by a Swiss National Science Foundation Grant (SNSF 31003A 159706/1), the Mäxi Foundation and GIF I-1289-412.13/2015 to O.M., and the Forschungskredit of the University of Zurich to Y.T. We thank the Center for Microscopy and Image Analysis at the University of Zurich (ZMB). R.D.G. was funded by National Institutes of Health grant GM106023 and the Progeria Research Foundation. We also thank K. H. Myung for her technical assistance, J. Rappaport of the Nikon Imaging Center in the Feinberg School of Medicine for support, and L. Chang of Nikon Instruments.

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Contributions

Y.T., R.D.G., K.T.S., M.E. and O.M. conceived and designed the experiments; Y.T. acquired the data and performed and analysed most of the experiments. M.E. analysed the structure of the lamin filaments, and evaluated the lamin gold-label distribution in the immunogold labelling experiments. T.S. and A.E.G. performed the 3D-SIM analysis. M.K. and K.B.H. acquired and analysed the data on the in vitro assembled lamin paracrystals, and A.D.G. refined the structural data within the manuscript revision process. O.M., Y.T., M.E. and K.T.S. discussed analysis, interpretations and presentation. Y.T. and O.M. wrote the manuscript with contributions from all of the authors.

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Correspondence to Ohad Medalia.

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Extended data figures and tables

Extended Data Figure 1 Sample preparation of vimentin deficient MEFs for cryo-ET and 3D-SIM.

a, The 3D-SIM images of immunolabelled wild-type (WT) and Vim−/− MEFs show similar localization and expression of lamin A and lamin B1, derived from n = 3 independent experiments. Scale bar, 5 μm. b, Western blot analyses of the indicated MEF cell lines show unchanged expression levels of each of the lamin isoforms and the retention of the lamins following the short hairpin RNA (shRNA)-mediated knockdown of vimentin. c, The 3D-SIM images of pre-permeabilized, nuclease treated and immunolabelled MEFs show similar localization and expression of lamin A and lamin B1 compared with untreated cells (a). Derived from n = 3 independent experiments. Scale bar, 1 μm. d, The localizations of the lamina-associated proteins (LAPs), emerin and Lap2β exhibit high similarities between pre-permeabilized and nuclease treated (+) and untreated (−) cells, as indicated by 3D-SIM analysis. Derived from n = 3 independent experiments. Scale bar, 1 μm. e, Illustration of the cryo-ET sample preparation procedure.

Extended Data Figure 2 Vitrified MEF nuclear envelopes on cryo-ET EM grids.

a, Low-magnification view of two nuclei (encircled by red lines) on a 100 μm × 100 μm carbon mesh of an EM-grid. Scale bar, 50 μm. b, Image shows a part of a nucleus (contoured by the light-grey line). Cryo-tomograms of 1.4 μm × 1.4 μm were acquired at various positions within the area indicated (1–4). Representative xy slices from sub-volumes containing filaments within regions 1–4 are shown in Fig. 1b. Scale bar, 2 μm. c, Higher-magnification projection view of a nucleus reveals some of the canonical components of the nucleus, for example a region of preserved nuclear double-membrane (white arrows), NPCs (white arrowheads) and a highly dense area with putative chromatin remnants (in the upper left corner). Scale bar, 1 μm. n = 55 for ac. d, Consecutive 1.36 nm xy slices show the thickness of the lamin meshwork and displays occasionally observed putative chromatin remnants (white arrows). Derived from n = 25 sub-volumes. Scale bar, 100 nm. e, Rendering shows segmented lamin filaments (yellow) and putative chromatin remnants (blue). This view of the segmented nuclear lamina displays individual filaments that cross each other at different positions along the z axis within a boundary of ~14 nm. Scale bar, 100 nm.

Extended Data Figure 3 Immunogold labelling of A- and B-type lamins.

Nuclei on EM grids were treated with anti-lamin A/C or anti-lamin B1 antibodies and labelled with 6 nm gold-conjugated protein A before vitrification and cryo-ET analysis. Control samples were treated with protein A conjugate only. a, Projection views of nuclei (contoured by the light-grey line) display the distribution of gold conjugate. Scale bar, 200 nm. b, Zoomed-in images show 9 nm thick xy slices through reconstructed volumes of the respective immunogold-labelled nuclei. A- and B-type filaments are labelled with gold conjugate as indicated (red circles). Scale bar, 100 nm. n = 24 for a and b. c, Gallery view of immunogold-labelled, segmented and skeletonized filaments from 40 nm thick sub-volumes. Green dots indicate immunogold-labelled lamin A and red dots lamin B1 in n = 9 sub-volumes each, locating both within sparsely and densely packed regions. Scale bar, 200 nm. d, Box plot shows the immunogold labelling density of lamin A/C and lamin B1 per μm2 (± s.d.) from volumes shown in c (white line represents the median and black dot the average number of gold particles).

Extended Data Figure 4 Co-immunogold labelling of A- and B-type lamins.

a, Nuclei on EM grids were treated with anti-lamin A/C and anti-lamin B1 and labelled with 6 nm and 10 nm gold conjugates, respectively, before vitrification and cryo-ET analysis, n = 24. Control samples (middle and right) were either treated with anti-lamin A/C before treatment with 6 nm gold conjugate, post-fixation and subsequent treatment with 10 nm gold conjugate (middle), or only with 6 nm and 10 nm gold conjugate (right), omitting incubation with antibodies. Large projection views of parts of nuclei (contoured by the light-grey line) display the distribution of gold conjugates under the indicated conditions. Scale bar, 200 nm. b, Box plots show the labelling density of 6 nm (lamin A/C) and 10 nm (lamin B1) gold particles per μm2 (± s.d.) under the indicated conditions (white line represents the median and black dot the average amount of gold particles). The number of gold particles was extracted from n = 47 sub-volumes (left), n = 9 sub-volumes (middle) or n = 11 sub-volumes (right). c, To validate the reliable assignment of small and large gold colloids, 6 nm and 10 nm gold-conjugated protein A labels were manually picked from samples containing either 6 nm or 10 nm colloids (individual gold colloids), a mixture of both (mixed gold colloids) or from the co-immunogold labelling experiment (lamin A/C and B1 co-labelling experiments) and averaged (n = 200 per condition). The two images at the bottom show a collage of nine individual 6 nm (left) and 10 nm (right) gold-conjugated protein A label, respectively, which were picked from the co-immunogold labelling experiments. The line plot shows the normalized intensity profile of the average diameter of the gold-conjugated protein A labels that were picked from the indicated samples. The solid lines show the intensity profile of the averaged gold label from the samples containing only one type of colloid. The dashed lines show the intensity profile of the averaged gold labels picked from a mixture. The lines with diamonds show the profile of the averaged gold labels from the co-immunogold labelling experiment. The line profiles of the 6 nm and 10 nm averaged gold-conjugated protein A labels are shown in green and red, respectively. The line plot shows almost identical diameters of the 6 nm or 10 nm gold labels for each condition.

Extended Data Figure 5 Lamin filament classification and averaging.

a, Gallery view illustrates a set of extracted and aligned filaments used for further analysis. b, Montage of 36 out of the 40 most populated class averages displays rod-like structures flanked by globular domains at different positions along the central rod. The yellow dots mark four of the most populated classes that are shown in Fig. 3a–c. The class index is given in the upper left corner of the sub-frames and the number of particles in the respective class in the lower left corner. c, Comparison of class averages with index 2 and 5 (Fig. 3c) with in vitro data17 shows remarkable similarities (yellow arrowheads).

Extended Data Figure 6 Structural analysis of in vitro assembled A- and B-type lamin paracrystals.

The final step in the in vitro assembly of assembled A- and B-type lamins results in the formation of paracrystals that display identical organization, corroborating that A- and B-type lamins assemble into similar structures a, TEM analysis and comparison of negatively stained human lamin C (adopted from ref. 41), B1 and B2 paracrystals show an identical striped pattern with 20 nm repeats. n = 20 paracrystals. Scale bar, 20 nm. b, Cryo-ET analysis of in vitro assembled lamin A shows the same 20 nm repeating pattern compared with the lamin isoforms shown in a; n = 9. Scale bar, 20 nm. c, The model shows the 2D arrangement of lamin protofilaments within a paracrystal. The rod-like structure is shown in grey and the globular tail domains in red. d, Averaged structure of in vitro assembled lamin A from cryo-tomograms, as shown in b, displays the striped pattern comprising the purported immunoglobulin-fold domains at distances of 20 nm, comparable to the spacing of the repeating pairs of globular domains in some structural classes from our in situ structural analysis (Fig. 3a, b). The distance of the putative immunoglobulin-fold domains within the stripes of the paracrystals is 6 nm. The averaged structure is derived from n = 4,370 sub-volumes. Scale bar, 20 nm.

Extended Data Figure 7 Lamin assembly scheme.

Averaging and classification of lamin filaments examined in situ show that lamin filaments are composed of two half-staggered head-to-tail polymers. For this, lamin dimers (left) assemble into dimeric head-to-tail polymers exhibiting short overlapping regions, tetrameric in a cross-section (middle). The immunoglobulin domains (red) along the head-to-tail polymer are positioned every ~40 nm. Ultimately, two head-to-tail polymers assemble laterally into a protofilament (right) with a uniformly shaped rod, ~3.5 nm in diameter, containing alternating tetra- and hexameric regions. In this assembly state the immunoglobulin domains are positioned every 20 nm alongside the lamin filament.

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Turgay, Y., Eibauer, M., Goldman, A. et al. The molecular architecture of lamins in somatic cells. Nature 543, 261–264 (2017). https://doi.org/10.1038/nature21382

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